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The Molecular Probes® Handbook A GUIDE TO FLUORESCENT PROBES AND LABELING TECHNOLOGIES 11th Edition (2010) Molecular Probes™ Handbook A Guide to Fluorescent Probes and Labeling Technologies 11th Edition (2010) CHAPTER 1 Fluorophores CHAPTER 13 and Their for Amine-Reactive Probes Lipids and Derivatives Membranes Molecular Probes Resources Molecular Probes Handbook (online version) Comprehensive guide to fluorescent probes and labeling technologies thermofisher.com/handbook Molecular Probes®SpectraViewer Resources Molecular Probes Fluorescence Identify compatible sets of fluorescent dyes and cell structure probes Molecular Probes® Handbook (online version) thermofisher.com/spectraviewer Comprehensive guide to fluorescent probes and labeling technologies lifetechnologies.com/handbook BioProbes Journal of Cell Biology Applications Award-winning magazine highlighting cell biology products and applications Fluorescence SpectraViewer thermofisher.com/bioprobes Identify compatible sets of fluorescent dyes and cell structure probes Access all Molecular Probes educational resources at thermofisher.com/probes lifetechnologies.com/spectraviewer BioProbes® Journal of Cell Biology Applications Award-winning magazine highlighting cell biology products and applications lifetechnologies.com/bioprobes Access all Molecular Probes® educational resources at lifetechnologies.com/mpeducat THIRTEEN CHAPTER 13 Probes for Lipids and Membranes 13.1 Introduction to Membrane Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547 Fluorescent and Biotinylated Membrane Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547 Fluorescent Analogs of Natural Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547 Other Lipophilic and Amphiphilic Fluorescent Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 548 Other Probes for Studying Cell Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 548 13.2 Fatty Acid Analogs and Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 549 Fluorescent Fatty Acid Analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 549 BODIPY® Fatty Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 549 NBD Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551 Pyrene Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551 Dansyl Undecanoic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551 cis-Parinaric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551 ADIFAB Fatty Acid Indicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553 Phospholipids with BODIPY® Dye–Labeled Acyl Chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 554 BODIPY® Glycerophospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 554 Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 556 Phospholipid with DPH-Labeled Acyl Chain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557 Properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557 Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557 Phospholipids with NBD-Labeled Acyl Chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557 Properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557 Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557 Phospholipids with Pyrene-Labeled Acyl Chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558 Properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558 Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558 Phospholipids with a Fluorescent or Biotinylated Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559 Phospholipid with a Dansyl-Labeled Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559 Phospholipid with a Marina Blue® Dye–Labeled Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559 Phospholipid with a Pacific Blue™ Dye–Labeled Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559 Phospholipid with an NBD-Labeled Head Group. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559 Phospholipid with a Fluorescein-Labeled Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559 Phospholipid with an Oregon Green® 488 Dye–Labeled Head Group. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560 Phospholipid with a BODIPY® FL Dye–Labeled Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560 Phospholipids with a Rhodamine or Texas Red® Dye–Labeled Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560 Phospholipids with a Biotinylated Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560 LipidTOX™ Phospholipid and Neutral Lipid Stains for High-Content Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561 HCS LipidTOX™ Phospholipidosis Detection Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561 HCS LipidTOX™ Neutral Lipid Stains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561 HCS LipidTOX™ Phospholipidosis and Steatosis Detection Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562 Data Table 13.2 Fatty Acid Analogs and Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563 Product List 13.2 Fatty Acid Analogs and Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes . . . . . . . . . . . . . . . . . . . . 566 Sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566 Structure and Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566 BODIPY® Sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567 ™ The Handbook: A Guide to Fluorescent Probes and Labeling Technologies TheMolecular MolecularProbes Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on IMPORTANT NOTICE Theand products manual are covered one orUse more Limited Use Label License(s). refer to theorAppendix page: 971 Master described Product Listinonthis page 975. Products are Forby Research Only. Not intended for any animal or Please human therapeutic diagnosticon use. page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes 545 Chapter 13 — Probes for Lipids and Membranes NBD Sphingolipids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568 Vybrant® Lipid Raft Labeling Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568 Amplex® Red Sphingomyelinase Assay Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 569 Steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 569 BODIPY® Cholesteryl Esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 569 Side Chain–Modified Cholesterol Analog . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570 Amplex® Red Cholesterol Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570 Fluorescent Triacylglycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571 Lipopolysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571 Fluorescent Lipopolysaccharides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571 Pro-Q® Emerald 300 Lipopolysaccharide Gel Stain Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572 Data Table 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573 Product List 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574 13.4 Dialkylcarbocyanine and Dialkylaminostyryl Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575 Dialkylcarbocyanine Probes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575 DiI, DiO, DiD, DiR and Analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575 Spectral Properties of Dialkylcarbocyanines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575 Substituted DiI and DiO Derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576 DiI and DiO as Probes of Membrane Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577 DiI and DiO as Probes of Membrane Dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577 Dialkylaminostyryl Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577 Data Table 13.4 Dialkylcarbocyanine and Dialkylaminostyryl Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 578 Product List 13.4 Dialkylcarbocyanine and Dialkylaminostyryl Probes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579 13.5 Other Nonpolar and Amphiphilic Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579 Amphiphilic Rhodamine, Fluorescein and Coumarin Derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579 Octadecyl Rhodamine B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579 Amphiphilic Fluoresceins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580 Amphiphilic Coumarin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580 DPH and DPH Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580 Diphenylhexatriene (DPH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580 TMA-DPH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581 Nonpolar BODIPY® Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581 BODIPY® Fluorophores. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581 BODIPY® FL C5-Ceramide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582 CellTrace™ BODIPY® TR Methyl Ester . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582 Pyrene, Nile Red and Bimane Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582 Nonpolar Pyrene Probe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582 Nile Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582 Bimane Azide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583 LipidTOX™ Neutral Lipid Stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583 Membrane Probes with Environment-Sensitive Spectral Shifts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584 Prodan and Laurdan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584 Dapoxyl® Derivative . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584 Anilinonaphthalenesulfonate (ANS) and Related Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584 Bis-ANS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585 DCVJ. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585 Data Table 13.5 Other Nonpolar and Amphiphilic Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 586 Product List 13.5 Other Nonpolar and Amphiphilic Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587 The Molecular Guide to to Fluorescent Fluorescent Probes The MolecularProbes® Probes Handbook: Handbook: AAGuide Probesand andLabeling LabelingTechnologies Technologies ™ 546 IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products in arethis For Research Usecovered Only. Notby intended anyLimited animal orUse human therapeutic or diagnostic use.to IMPORTANT NOTICE : The products described manual are one or for more Label License(s). Please refer the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes Chapter 13 — Probes for Lipids and Membranes Section 13.1 Introduction to Membrane Probes 13.1 Introduction to Membrane Probes Fluorescent and Biotinylated Membrane Probes The plasma membranes and intracellular membranes of live cells and the artificial membranes of liposomes represent a significant area of application for fluorescent probes. Membrane probes include fluorescent analogs of natural lipids, as well as lipophilic organic dyes that have little structural resemblance to natural biomolecules. We offer a wide range of both types of membrane probes. These probes are used for structural and biophysical analysis of membranes, for following lipid transport and metabolism in live cells (Figure 13.1.1) and for investigating synaptosome recycling (Section 16.1) and lipid-mediated signal transduction processes (Chapter 17). Due to their low toxicity and stable retention, some lipid probes are particularly useful for long-term cell tracing (Section 14.4). Other, slightly less lipophilic probes are used as membrane markers of endocytosis and exocytosis (Section 16.1). Fluorescent Analogs of Natural Lipids We offer fluorescent and, in a few cases, biotinylated analogs of five naturally occurring lipid classes—phospholipids, sphingolipids (including ceramides), fatty acids, triglycerides and steroids. Phospholipids are the principal building blocks of cell membranes. Most phospholipids are derivatives of glycerol comprising two fatty acyl residues (nonpolar tails) and a single phosphate ester substituent (polar head group). Despite their overall structural similarity (Figure 13.1.2), natural phospholipids exhibit subtle differences in their fatty acid compositions, degree of acyl A HOCH + (CH ) NCH CH O 33 2 2 O CH CH OH OH 2 Glycerol O P 2 Figure 13.1.1 The cytoplasm of a live zebrafish embryo labeled with the green-fluorescent lipophilic tracer BODIPY® 505/515 (D3921). The image was contributed by Arantza Barrios, University College, London. - O CH CH CH O O O C C R R 2 2 O O P O - O CH O Phosphatidylcholine O OH O P O CH 2 O OH OH HO OH CH CH O O O C C R R CH CH O O O C C R R 2 2 O Phosphatidic Acid 2 O Phosphatidylinositol B HOCH 2 CH NH + (CH ) NCH CH O 33 2 2 CH 2 O CH(CH ) CH 2 12 3 Sphingosine O P CH OH - O CH 2 CH CH NH OH C CH HO CH(CH ) CH 2 12 3 OH O HO R Sphingomyelin O O CH 2 2 CH CH NH OH C CH CH(CH ) CH 2 12 3 CH NH OH C OH HOCH CH CH CH(CH ) CH 2 12 3 O R Cerebroside O R Ceramide Figure 13.1.2 A) Phosphatidylcholines, phosphatidylinositols and phosphatidic acids are examples of glycerolipids derived from glycerol. B) Sphingomyelins, ceramides and cerebrosides are examples of sphingolipids derived from sphingosine. In all the structures shown, R represents the hydrocarbon tail portion of a fatty acid residue. ™ The Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies TheMolecular Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPORTANT NOTICE:described The products described thiscovered manual are by oneLimited or moreUse Limited Use Label License(s). to the Appendix IMPORTANT NOTICE : The products in this manualinare by covered one or more Label License(s). PleasePlease referrefer to the Appendix on on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes 547 Chapter 13 — Probes for Lipids and Membranes Section 13.1 Introduction to Membrane Probes chain unsaturation and type of polar head group.1 These differences can produce significant variations in membrane physical properties, in the location of phospholipids in a lipid bilayer and in their biological activity. Fluorescent phospholipid analogs (Section 13.2) can be classified according to where the fluorophore is attached. The fluorophore can be attached to one (or both) of the fatty acyl chains or to the polar head group. The attachment position of the fluorophore determines whether it is located in the nonpolar interior or at the water/lipid interface when the phospholipid analog is incorporated into a lipid bilayer membrane. Fatty acids are the building blocks for a diverse set of biomolecules. Some fatty acids (e.g., arachidonic acid) are important in cell signaling.2 Fatty acids are liberated by the enzymatic action of phospholipase A on phospholipids (Section 17.4) and also by various other lipases. Fluorescent fatty acids can often be used interchangeably with the corresponding phospholipids as membrane probes; however, fatty acids transfer more readily between aqueous and lipid phases.3 Although fatty acids are ionized at neutral pH in water (pKa ~5), their pKa is typically ~7 in membranes, and thus a significant fraction of membrane-bound fatty acids are neutral species.3 Certain fluorescent fatty acids (Section 13.2) are readily metabolized by live cells to phospholipids, mono-, diand triacylglycerols, cholesteryl esters and other lipid derivatives.4 Sphingolipids play critical roles in processes such as proliferation, apoptosis, signal transduction and molecular recognition at cell surfaces.1,5,6 Defects in the lysosomal breakdown of sphingolipids are the underlying cause of lipid storage disorders such as Niemann–Pick, Tay– Sachs, Krabbe and Gaucher diseases. The sphingolipids described in Section 13.3 include ceramides, sphingomyelins, glycosylceramides (cerebrosides) and gangliosides. The structural backbone of sphingolipids is the lipophilic amino–dialcohol sphingosine (2-amino-4-octadecen1,3-diol, Figure 13.1.2) to which a single fatty acid residue is attached via an amide linkage. Our fluorescent analogs of sphingolipids are prepared by replacing the natural amide-linked fatty acid with a fluorescent analog. Sphingolipids with an unmodified hydroxyl group in the 1-position are classified as ceramides. As part of the lipid-sorting process in cells, ceramides are glycosylated to cerebrosides (Figure 13.1.2) or converted to sphingomyelins (Figure 13.1.2) in the Golgi complex. Glycosylated sphingolipids (cerebrosides and gangliosides) occur in the plasma membranes of all eukaryotic cells and are involved in cell recognition, signal transduction and modulation of receptor function.7 Gangliosides have complex oligosaccharide head groups containing at least one sialic acid residue in place of the single galactose or glucose residues of cerebrosides. Fluorescent cholesteryl esters and triglycerides (Section 13.3) can be used as structural probes and transport markers for these important lipid constituents of membranes and lipoproteins.8 They may also serve as fluorescent substrates for lipases and lipid-transfer proteins and can be incorporated into low-density lipoproteins (LDL, Section 16.1). Other Lipophilic and Amphiphilic Fluorescent Probes The probes described in Section 13.4 and Section 13.5 are not analogs of any particular biological lipid class, but they have a general structural resemblance that facilitates labeling of membranes, lipoproteins and other lipid-based molecular assemblies. Particularly notable members of this group are the lipophilic carbocyanines DiI (Figure 13.1.3), DiO, DiD and DiR, the lipid fluidity probes DPH and TMA-DPH and the membrane-surface probes ANS and laurdan. These probes generally have limited water solubility and exhibit substantially enhanced fluorescence upon partition into lipid environments. They can be classified as either amphiphilic (having both polar and nonpolar structural elements) or neutral (lacking charges and most soluble in very nonpolar environments). We use similar neutral lipophilic dyes for internal staining of our fluorescent polystyrene microspheres (Section 6.5). Other Probes for Studying Cell Membranes In addition to the lipophilic probes described in this chapter, we have available the following products for studying the properties and functions of cell membranes: • Moderately lipophilic stains for the endoplasmic reticulum and Golgi apparatus (Section 12.4) • FM® dyes—amphiphilic probes for cell membrane labeling (Section 14.4, Section 16.1) • CellLight® Plasma Membrane-CFP, CellLight® Plasma MembraneGFP and CellLight® Plasma Membrane-RFP, which are BacMam 2.0 vectors encoding fluorescent proteins targeted to the plasma membrane (C10606, C10607, C10608; Section 14.4) • Alexa Fluor® dye–labeled cholera toxin subunit B conjugates for labeling lipid rafts (Section 14.7) • Annexin V conjugates for detection of phosphatidylserine exposure in apoptotic cell membranes (Section 15.5) • Fluorescent and fluorogenic phospholipase A substrates (Section 17.4) • Amplex® Red Phosphatidylcholine-Specific Phospholipase C Assay Kit and Amplex® Red Phospholipase D Assay Kit (A12218, A12219; Section 17.4) • Antibodies to phosphatidylinositol phosphates (Section 17.4) • Lipophilic pH indicators (Section 20.4) • Membrane potential–sensitive probes (Section 22.2, Section 22.3) REFERENCES Figure 13.1.3 The neuronal tracer DiI (D282, D3911) used as a diagnostic tool to evaluate patterns of innervation in newborn mouse cochlea. The larger image is of a mutant cochlea and the inset is of a wild-type cochlea. Image contributed by Bernd Fritzsch, Creighton University, and L. Reichardt and I. Farinas, Howard Hughes Medical Institute, San Francisco. 1. Nat Rev Mol Cell Biol (2008) 9:112; 2. Biochim Biophys Acta (1994) 1212:26; 3. J Lipid Res (1998) 39:467; 4. Chem Phys Lipids (1991) 58:111; 5. Annu Rev Biochem (1998) 67:27; 6. Biochim Biophys Acta (1991) 1082:113; 7. Ann N Y Acad Sci (1998) 845:57; 8. Nat Rev Mol Cell Biol (2008) 9:125. The MolecularProbes® Probes Handbook: Handbook: AAGuide Probes and and Labeling LabelingTechnologies Technologies The Molecular Guideto toFluorescent Fluorescent Probes ™ 548 IMPORTANT NOTICE: The products described in this manual coveredare by covered one or more Limited Use Label License(s). Please refer to thePlease Appendix onto IMPORTANT NOTICE : The products described in thisaremanual by one or more Limited Use Label License(s). refer page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes Chapter 13 — Probes for Lipids and Membranes Section 13.2 Fatty Acid Analogs and Phospholipids 13.2 Fatty Acid Analogs and Phospholipids The probes in this section and in Section 13.3 bear some structural resemblance to natural lipids. Included in this section are fluorescent fatty acid analogs, as well as phospholipids wherein one or both fatty acid esters are replaced by fluorescent fatty acid esters. The fluorophores in these probes tend to remain buried in the hydrophobic interior of lipid bilayer membranes.1,2 In this location, they are sensitive to membrane properties such as lipid fluidity, lateral domain formation and structural perturbation by proteins, drugs and other additives. Also included in this section are several head group–modified phospholipid analogs incorporating a fluorophore or biotin (Table 13.1). Sphingolipids, steroids and lipopolysaccharides are discussed in Section 13.3. Important applications of the fluorescent phosphatidylinositol derivatives as probes for signal transduction and various fluorescent phospholipids as phospholipase substrates are further described in Section 17.4. A review of fluorescent lipid probes and their use in biological and biophysical research has been published.3 Fluorescent Fatty Acid Analogs Our fluorescent fatty acid analogs have a fluorophore linked within the fatty acid chain or, more commonly, at the terminal (omega) carbon atom that is furthest from the carboxylate moiety. Although fluorescent fatty acid analogs are sometimes used as direct probes for membranes and liposomes, their most common applications have been for synthesis of fluorescent phospholipids and for metabolic incorporation by live cells. Our fluorescent fatty acids currently include derivatives based on the BODIPY®, nitrobenzoxadiazole (NBD), pyrene and dansyl fluorophores, as well as the naturally fluorescent polyunsaturated fatty acid, cis-parinaric acid. BODIPY® Fatty Acids BODIPY® fatty acids are, by far, the most fluorescent fatty acid analogs that we have available.4 The lack of ionic charge on the BODIPY® fluorophore is unusual for long-wavelength fluorescent dyes and results in exclusive localization of the fluorophore within the membrane (Figure 13.2.1). BODIPY® derivatives typically have extinction coefficients greater than 90,000 cm–1M–1 with absorption maxima beyond 500 nm. A useful spectroscopic property of BODIPY® dyes is the concentration-dependent formation of excited-state dimers ("excimers") with red-shifted emission. We have observed this phenomenon particularly with our green-fluorescent BODIPY® fatty acid derivatives, which undergo a considerable red shift in their emission when metabolically incorporated into lipophilic products 5 (Figure 13.2.2). Pyrene A E O C O - O N O 2 N N NH O B C O F O F N B F C O _ N G O Figure 13.2.2 BHK cells incubated in medium containing the fluorescent fatty acid analog BODIPY® 500/510 C1,C12 (D3823). This photomicrograph, obtained through a standard fluorescein longpass filter set, reveals reticular green-fluorescent staining as well as yelloworange–fluorescent spherical structures. These fluorescent structures are indicative of the metabolic accumulation of BODIPY® dye–labeled neutral lipids in cytoplasmic droplets. Image contributed by Juha Kasurinen, University of Helsinki, Finland. O + N( CH CH ) 2 3 2 O ( CH CH ) N 3 2 2 C C O SO 3 C _ O SO 2 NH Table 13.1 Phospholipids with labeled head groups. Label (Ex/Em) * Dansyl (336/517) Marina Blue® (365/460) Pacific Blue™ (410/455) NBD (463/536) Fluorescein (496/519) Oregon Green® 488 (501/526) BODIPY® FL (505/511) BODIPY® 530/550 Tetramethylrhodamine (540/566) Lissamine rhodamine (560/581) Texas Red® (582/601) Biotin (<250/none) Cat. No. D57 M12652 P22652 N360 F362 O12650 D3800 D3815 T1391 L1392 T1395MP B1550, B1616 * Fluorescence excitation (Ex) and emission (Em) spectral maxima, in nm, are in methanol. The spectra may be different in membranes. H D H C 3 N + N CH 3 N H C 3 CH CH _ H C 3 O O CH H C 3 N O + _ C O O C I NH O Figure 13.2.1 Location and orientation of representative fluorescent membrane probes in a phospholipid bilayer: A) DPH (D202), B) NBD-C6-HPC (N3786), C) bis-pyrene-PC (B3782), D) DiI (D282), E) cis-parinaric acid (P36005), F) BODIPY® 500/510 C4, C9 (B3824), G) N-Rh-PE (L1392), H) DiA (D3883) and I) C12-fluorescein (D109). ™ The Handbook: A Guide to Fluorescent Probes and Labeling TheMolecular MolecularProbes Probes® Handbook: A Guide to Fluorescent Probes and LabelingTechnologies Technologies IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes 549 Chapter 13 — Probes for Lipids and Membranes Section 13.2 Fatty Acid Analogs and Phospholipids H3C N H3 C F B N O F (CH2)15 C OH Figure 13.2.3 4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid (BODIPY® FL C16, D3821). O N F B N C F OH C -BODIPY® 500/510 C12 1 N F B N O F C OH C -BODIPY® 500/510 C9 4 N F B N O F C OH C -BODIPY® 500/510 C5 8 Figure 13.2.4 Structural representations showing the positional shift of the fluorophore with respect to the terminal carboxyl group in a homologous series of BODIPY® 500/510 fatty acids (D3823, B3824, D3825). N F B N O F (CH2)11 C OH Figure 13.2.5 4,4-Difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (BODIPY® 530/550 C12, D3832). N F B N fatty acids also exhibit excimer formation but their emission is at much shorter wavelengths than that of the BODIPY® dyes and they are therefore less suitable for the study of live cells. The fluorophores in our current selection of BODIPY® fatty acids and their approximate absorption/emission maxima (in nm) are: • BODIPY® 503/512 (BODIPY® FL; D3821, Figure 13.2.3; D3822; D3834; D3862) • BODIPY® 500/510 (D3823, B3824, D3825; Figure 13.2.4) • BODIPY® 530/550 (D3832, Figure 13.2.5) • BODIPY® 558/568 (D3835, Figure 13.2.6) • BODIPY® 581/591 (D3861) BODIPY® fatty acids are synthetic precursors to a wide variety of fluorescent phospholipids (described below), as well as several important sphingolipid probes described in Section 13.3. Some BODIPY® fatty acids are readily metabolized by live cells to phospholipids, di- and triacylglycerols, cholesteryl esters and other natural lipids.6–9 Analysis of cellular lipid extracts by HPLC has shown that glycerophosphocholines constitute more than 90% of the products of biosynthetic incorporation of BODIPY® 500/510 dodecanoic acid (D3823) by BHK cells.5 The three BODIPY® 500/510 probes form a unique series in which the green-fluorescent fluorophore is located within the fatty acid chain at different distances from the terminal carboxylate group.4 The overall length of the probe is constant and, including the fluorophore, is about equivalent to that of an 18-carbon fatty acid (Figure 13.2.4). BODIPY® 581/591 undecanoic acid (D3861) is particularly useful for detecting reactive oxygen species in cells and membranes.10–13 Oxidation of the polyunsaturated butadienyl portion of the BODIPY® 581/591 dye (Figure 13.2.7) truncates the conjugated π-electron system, resulting in a shift of the fluorescence emission peak from ~590 nm to ~510 nm.10,13,14 This oxidation response mechanism is similar to that of the naturally occurring polyunsaturated fatty acid cis-parinaric acid. In comparison to cis-parinaric acid, advantages of BODIPY® 581/591 undecanoic acid include: • Long-wavelength excitation, compatible with confocal laser-scanning microscopes and flow cytometers • Avoidance of photooxidation effects induced by ultraviolet excitation • Less interference by colored oxidant and antioxidant additives when detecting probe fluorescence 12 • Greater resistance to spontaneous oxidation • Red-to-green fluorescence shift, allowing the use of fluorescence ratio detection methods 10,13 O F (CH2)11 � C OH Figure 13.2.6 4,4-Difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (BODIPY® 558/568 C12, D3835). Figure 13.2.7 4,4-Difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3undecanoic acid (BODIPY® 581/591 C11, D3861). TheMolecular MolecularProbes® Probes Handbook: Handbook: AAGuide and Labeling LabelingTechnologies Technologies The Guideto toFluorescent Fluorescent Probes Probes and ™ 550 IMPORTANT NOTICE: The products described in this manual covered bycovered one or more Limited Use Label License(s). Please refer to thePlease Appendix IMPORTANT NOTICE : The products described in thisare manual are by one or more Limited Use Label License(s). referonto page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes Chapter 13 — Probes for Lipids and Membranes Section 13.2 Fatty Acid Analogs and Phospholipids O An alternative technique for detecting lipid peroxidation utilizes the oxidation-induced decrease of concentration-dependent excimer formation by BODIPY® FL dye–labeled fatty acids.15 NH(CH2)5 N NBD Fatty Acids Fluorescence of the nitrobenzoxadiazole (NBD) fluorophore is highly sensitive to its environment. Although it is moderately fluorescent in aprotic solvents, in aqueous solvents it is almost nonfluorescent.16 The NBD fluorophore is moderately polar and both its homologous 6-carbon and 12-carbon fatty acid analogs (N316, Figure 13.2.8; N678) and the phospholipids derived from these probes (N3786, N3787) tend to sense the lipid–water interface region of membranes instead of the hydrophobic interior 17 (see part B of Figure 13.2.1). The environmental sensitivity of NBD fatty acids can be usefully exploited to probe the ligand-binding sites of fatty acid and sterol carrier proteins.18 NBD fatty acids are not well metabolized by live cells.9,19 N C OH O NO2 Figure 13.2.8 NBD-X (6-(N-(7-nitrobenz-2-oxa-1,3-diazol4-yl)amino)hexanoic acid; N316). Pyrene Fatty Acids Figure 13.2.9 1-Pyrenedodecanoic acid (P96). Fluorescence emission The hydrophobic pyrene fluorophore is readily accommodated within the membrane.20 ω-Pyrene derivatives of longer-chain fatty acids (Figure 13.2.9) were first described by Galla and Sackmann in 1975.21 We offer pyrene derivatives of the 4-, 10-, 12- and 16-carbon fatty acids (P1903MP, P31, P96, P243, respectively). Pyrenebutanoic acid—frequently called pyrenebutyric acid (P1903MP)—has rarely been used as a membrane probe; however, its conjugates have exceptionally long excited-state lifetimes (τ >100 nanoseconds) and are consequently useful for time-resolved fluorescence immunoassays and nucleic acid detection.22,23 The long excited-state lifetime of pyrenebutyric acid also makes it useful as a probe for oxygen in cells 24–27 and lipid vesicles.28 Pyrene derivatives form excited-state dimers (excimers) with red-shifted fluorescence emission 29–31 (Figure 13.2.10). Pyrene excimers can even form when two pyrenes are tethered by a short trimethine spacer, as in 1,3-bis-(1-pyrenyl)propane (B311, Section 13.5). Pyrene excimer formation is commonly exploited for assaying membrane fusion 32,33 (Lipid-Mixing Assays of Membrane Fusion—Note 13.1) and for detecting lipid domain formation. 34–36 Pyrene fatty acids are metabolically incorporated into phospholipids, di- and tri-acylglycerols and cholesteryl esters by live cells.19,37,38 Other uses of pyrene fatty acids include: 1 2 3 4 350 • • • • • Detecting lipid–protein interactions 9,40 Inducing photodynamic damage 41,42 Investigating phospholipase A 2 action on lipid assemblies 43–45 Studying lipid transport mechanisms and transfer proteins 46–48 Synthesizing fluorescent sphingolipid probes 49–52 Dansyl Undecanoic Acid Dansyl undecanoic acid (DAUDA, D94; Figure 13.2.11) incorporates a polar, environmentsensitive dansyl fluorophore that preferentially locates in the polar headgroup region of lipid bilayer membranes.53 DAUDA exhibits a 60-fold fluorescence enhancement and a large emission spectral shift to shorter wavelengths on binding to certain proteins.54 This property has been exploited to analyze fatty acid–binding proteins 54–57 and also to develop a fluorometric phospholipase A 2 assay (Section 17.4) based on competitive fatty acid displacement.58–61 400 450 500 550 600 Wavelength (nm) Figure 13.2.10 Excimer formation by pyrene in ethanol. Spectra are normalized to the 371.5 nm peak of the monomer. All spectra are essentially identical below 400 nm after normalization. Spectra are as follows: 1) 2 mM pyrene, purged with argon to remove oxygen; 2) 2 mM pyrene, airequilibrated; 3) 0.5 mM pyrene (argon-purged); and 4) 2 µM pyrene (argon-purged). The monomer-to-excimer ratio (371.5 nm/470 nm) is dependent on both pyrene concentration and the excited-state lifetime, which is variable because of quenching by oxygen. cis-Parinaric Acid The naturally occurring polyunsaturated fatty acid cis-parinaric acid (P36005, Figure 13.2.12) was initially developed as a membrane probe by Hudson and co-workers and published in 1975.62 cis-Parinaric acid is the closest structural analog of intrinsic membrane lipids among currently available fluorescent probes (Figure 13.2.1). The chemical and physical properties of cis-parinaric acid have been well characterized. The lowest absorption band of cis-parinaric acid has two main peaks around 300 nm and 320 nm, with a high extinction coefficient. cis-Parinaric acid offers several experimentally advantageous optical properties, including a very large fluorescence Stokes shift (~100 nm) and an almost complete lack of fluorescence in water. In addition, the fluorescence decay lifetime of cis-parinaric acid varies from 1 to ~40 nanoseconds, depending on the molecular packing density in phospholipid bilayers. Consequently, minutely detailed information on lipid-bilayer dynamics can be obtained. Figure 13.2.11 11-((5-Dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid (DAUDA, D94). H CH3CH2 C C H H C C H H C C H H C C O (CH2)� C OH H Figure 13.2.12 cis-Parinaric acid (P36005). ™ The Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies TheMolecular Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPORTANT NOTICE:described The products described thiscovered manual are by oneLimited or moreUse Limited Use Label License(s). to the Appendix IMPORTANT NOTICE : The products in this manualinare by covered one or more Label License(s). PleasePlease referrefer to the Appendix on on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes 551 Chapter 13 — Probes for Lipids and Membranes Section 13.2 Fatty Acid Analogs and Phospholipids NOTE 13.1 Lipid-Mixing Assays of Membrane Fusion Fluorometric methods for assaying membrane fusion exploit processes, such as nonradiative energy transfer, fluorescence quenching and pyrene excimer formation, that are dependent on probe concentration.1–8 Assays of membrane fusion report either the mixing of membrane lipids (described here) or the mixing of the aqueous contents of the fused entities (Assays of Volume Change, Membrane Fusion and Membrane Permeability—Note 14.3). Chapter 13 describes additional methods for detecting membrane fusion based on image analysis. NBD–Rhodamine Energy Transfer Principle: Struck, Hoekstra and Pagano introduced lipid-mixing assays based on NBD–rhodamine energy transfer.9 In this method (Figure 1), membranes labeled with a combination of fluorescence energy transfer donor and acceptor lipid probes—typically NBD-PE and N-Rh-PE (N360, L1392; Section 13.2), respectively—are mixed with unlabeled membranes. Fluorescence resonance energy transfer (FRET), detected as rhodamine emission at ~585 nm resulting from NBD excitation at ~470 nm, decreases when the average spatial separation of the probes is increased upon fusion of labeled membranes with unlabeled membranes. The reverse detection scheme, in which FRET increases upon fusion of membranes that have been separately labeled with donor and acceptor probes, has also proven to be a useful lipid-mixing assay.10 Applications: Applications of the NBD–rhodamine assay are described in footnoted references.11–20 Figure 1 Pictorial representation of a lipid-mixing assay based on fluorescence resonance energy transfer (FRET). The average spatial separation of the donor (D) and acceptor (A) lipid probes increases upon fusion of labeled membranes with unlabeled membranes, resulting in decreased efficiency of proximity-dependent FRET (represented by yellow arrows). Decreased FRET efficiency is registered by increased donor fluorescence intensity and decreased acceptor fluorescence intensity. Octadecyl Rhodamine B Self-Quenching Principle: Lipid-mixing assays based on self-quenching of octadecyl rhodamine B (R18, O246; Section 13.5) were originally described by Hoekstra and co-workers.21 Octadecyl rhodamine B self-quenching occurs when the probe is incorporated into membrane lipids at concentrations of 1–10 mole percent.22 Unlike phospholipid analogs, octadecyl rhodamine B can readily be introduced into existing membranes in large amounts. Fusion with unlabeled membranes results in dilution of the probe, which is accompanied by increasing fluorescence 23,24 (excitation/emission maxima 560/590 nm) (Figure 2). The assay may be compromised by effects such as spontaneous transfer of the probe to unlabeled membranes, quenching of fluorescence by proteins and probe-related inactivation of viruses; the prevalence of these effects is currently debated.25–27 Applications: The octadecyl rhodamine B self-quenching assay is extensively used for detecting virus–cell fusion.28–39 Pyrene Excimer Formation Principle: Pyrene-labeled fatty acids (e.g., P31, P96, P243; Section 13.2) can be biosynthetically incorporated into viruses and cells in sufficient quantities to produce the degree of labeling required for long-wavelength pyrene excimer fluorescence (Figure 3). This excimer fluorescence is diminished upon fusion of labeled membranes with unlabeled membranes (Figure 4). Fusion can be monitored by following the increase in the ratio of monomer (~400 nm) to excimer (~470 nm) emission, with excitation at about 340 nm. This method appears to circumvent some of the potential artifacts of the octadecyl rhodamine B self-quenching technique 26 and, therefore, provides a useful alternative for virus–cell fusion applications. Applications: Applications of pyrene excimer assays for membrane fusion are described in the footnoted references.26,28,40–43 Figure 2 Pictorial representation of a lipid-mixing assay based on fluorescence self-quenching. Fluorescence of octadecyl rhodamine B (O246), incorporated at >1:100 with respect to host membrane lipids, is quenched due to dye–dye interactions. Fusion with unlabeled membranes causes dispersion of the probe, resulting in a fluorescence increase that is represented here by a color change from black to green. O O O O O OO + Pyrene excimer fluorescence ~470 nm O O O O O O O Fusion Pyrene monomer fluorescence ~400 nm Figure 4 Pictorial representation of a lipid-mixing assay based on pyrene excimer formation. Locally concentrated pyrene-labeled lipid probes emit red-shifted fluorescence due to formation of excimers (excited-state dimers). Probe dilution by unlabeled lipids as a result of membrane fusion is registered by the replacement of excimer emission by blue-shifted monomer fluorescence. The MolecularProbes® Probes Handbook: Handbook: AA Guide Probesand andLabeling LabelingTechnologies Technologies The Molecular Guide to to Fluorescent Fluorescent Probes ™ 552 O O IMPORTANT NOTICE: The products described in this manual coveredare by covered one or more Limited Use Label License(s). Please refer to thePlease Appendix onto IMPORTANT NOTICE : The products described in thisaremanual by one or more Limited Use Label License(s). refer page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes Chapter 13 — Probes for Lipids and Membranes Section 13.2 Fatty Acid Analogs and Phospholipids • Measurement of peroxidation in lipoproteins 63–65 and the relationship of peroxidation to cytotoxicity 66,67 and apoptosis 68–71 • Evaluation of antioxidants 72–75 • Detection of lipoproteins following chromatographic separation 76 and structural characterization of lipoproteins 77 • Detection of lipid–protein interactions 78–80 and lipid clustering 81 • High-affinity binding to a hydrophobic pocket between the heavy chain of myosin subfragment-1 and its essential light chain 82 • Investigation of the mechanism of fatty acid–binding proteins 83–85 and phospholipid-transfer proteins 86,87 1 2 3 4 350 400 450 500 550 600 Wavelength (nm) Figure 3 Excimer formation by pyrene in ethanol. Spectra are normalized to the 371.5 nm peak of the monomer. All spectra are essentially identical below 400 nm after normalization. Spectra are as follows: 1) 2 mM pyrene, purged with argon to remove oxygen; 2) 2 mM pyrene, air-equilibrated; 3) 0.5 mM pyrene (argon-purged); and 4) 2 µM pyrene (argon-purged). The monomer-to-excimer ratio (371.5 nm/470 nm) is dependent on both pyrene concentration and the excited-state lifetime, which is variable because of quenching by oxygen. 1. Chem Phys Lipids (2002) 116:39; 2. Anal Biochem (2009) 386:91; 3. Methods Enzymol (1993) 220:3; 4. Methods Enzymol (1993) 220:15; 5. Proc Natl Acad Sci U S A (2009) 106:979; 6. Annu Rev Biophys Biophys Chem (1989) 18:187; 7. Hepatology (1990) 12:61S-66S; 8. Biochemistry (1987) 26:8435; 9. Biochemistry (1981) 20:4093; 10. Methods Enzymol (1993) 221:239; 11. Biochemistry (1994) 33:12615; 12. Biochemistry (1994) 33:5805; 13. Biochemistry (1994) 33:3201; 14. Biophys J (1994) 67:1117; 15. J Biol Chem (1994) 269:15124; 16. J Biol Chem (1994) 269:4050; 17. J Biol Chem (1993) 268:1716; 18. Biochemistry (1992) 31:2629; 19. Biochemistry (1991) 30:5319; 20. J Biol Chem (1991) 266:3252; 21. Biochemistry (1984) 23:5675; 22. J Biol Chem (1990) 265:13533; 23. Biophys J (1993) 65:325; 24. Biophys J (1990) 58:1157; 25. Biochim Biophys Acta (1994) 1190:360; 26. Biochemistry (1993) 32:11330; 27. Biochemistry (1993) 32:900; 28. Biochemistry (1994) 33:9110; 29. Biochemistry (1994) 33:1977; 30. Biochim Biophys Acta (1994) 1191:375; 31. J Biol Chem (1994) 269:5467; 32. Biochem J (1993) 294:325; 33. J Biol Chem (1993) 268:25764; 34. J Biol Chem (1993) 268:9267; 35. Virology (1993) 195:855; 36. Biochemistry (1992) 31:10108; 37. Exp Cell Res (1991) 195:137; 38. J Virol (1991) 65:4063; 39. Biochemistry (1990) 29:4054; 40. EMBO J (1993) 12:693; 41. J Virol (1992) 66:7309; 42. Biochemistry (1988) 27:30; 43. Biochim Biophys Acta (1986) 860:301. The extensive unsaturation of cis-parinaric acid makes it quite susceptible to oxidation. Consequently, we offer cis-parinaric acid in a 10 mL unit size of a 3 mM solution in deoxygenated ethanol (P36005); if stored protected from light under an inert argon atmosphere at –20°C, this stock solution should be stable for at least six months. During experiments, we strongly advise handling cis-parinaric acid samples under inert gas and preparing solutions using degassed buffers and solvents. cis-Parinaric acid is also somewhat photolabile, undergoing photodimerization under intense illumination, resulting in loss of fluorescence. 88 ADIFAB Fatty Acid Indicator Fatty acid–binding proteins are small cytosolic proteins found in a variety of mammalian tissues, and studies of their physiological function frequently involve fluorescent fatty acid probes.89 To facilitate these studies, we offer ADIFAB reagent (A3880), a dual-wavelength fluorescent indicator of free fatty acids 90–92 (Figure 13.2.13, Figure 13.2.14). ADIFAB reagent is a conjugate of I-FABP, a rat intestinal fatty acid–binding protein with a low molecular weight (15,000 daltons) and a high binding affinity for free fatty acids,93 and the polarity-sensitive acrylodan fluorophore (A433, Section 2.3). It is designed to provide quantitative monitoring of free fatty acids without resorting to separative biochemical methods.44,94,95 With appropriate precautions, which are described in the product information sheet accompanying this product, ADIFAB can be used to determine free fatty acid concentrations between 1 nM and >20 µM. Ex = 390 nm _OA Fluorescence emission Fluorescence emission Selected applications of cis-parinaric acid include: 400 +OA 450 500 550 600 650 Wavelength (nm) Figure 13.2.13 Ribbon representation of the ADIFAB free fatty acid indicator (A3880). In the left-hand image, the fatty acid binding site of intestinal fatty acid–binding protein (yellow) is occupied by a covalently attached acrylodan fluorophore (blue). In the right-hand image, a fatty acid molecule (gray) binds to the protein, displacing the fluorophore (green) and producing a shift of its fluorescence emission spectrum. Image contributed by Alan Kleinfeld, FFA Sciences LLC, San Diego. Figure 13.2.14 The free fatty acid–dependent spectral shift of ADIFAB (A3880). Spectra shown represent 0.2 µM ADIFAB in pH 8.0 buffer with (+OA) and without (–OA) addition of 4.7 µM cis9-octadecenoic (oleic) acid (OA). The ratio of fluorescence emission intensities at 505 nm and 432 nm can be quantitatively related to free fatty acid concentrations. ™ The Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies TheMolecular Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPORTANT NOTICE:described The products described thiscovered manual are by one or moreUse Limited Label License(s). to Appendix the Appendix IMPORTANT NOTICE : The products in this manualinare by covered one or more Limited LabelUse License(s). PleasePlease refer refer to the on on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes 553 Chapter 13 — Probes for Lipids and Membranes Section 13.2 Fatty Acid Analogs and Phospholipids Phospholipids with BODIPY® Dye–Labeled Acyl Chains BODIPY® Glycerophospholipids Figure 13.2.15 2-(4,4-Difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (β-BODIPY® 500/510 C12-HPC, D3793). O CH3(CH2)1� C OCH2 H3C F N B (CH2)11 C OCH F O N O CH2O � OCH2CH2N(CH3)3 O H3C Figure 13.2.16 2-(4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (β-BODIPY® FL C12-HPC, D3792). O CH3(CH2)1� C OCH2 H3C F N B (CH2)� F N C OCH O O CH2O � OCH2CH2N(CH3)3 O H3C Figure 13.2.17 2-(4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (β-BODIPY® FL C5-HPC, D3803). O CH3(CH2)1� C OCH2 F N B (CH2)� F N C OCH O O CH2O � OCH2CH2N(CH3)3 O Figure 13.2.18 2-(4,4-Difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (β-BODIPY® 530/550 C5-HPC, D3815). O CH3(CH2)1� C OCH2 H3C F N B F (CH2)� C OCH O N O CH2O � O O 2�NH� H3C Figure 13.2.19 2-(4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphate, diammonium salt (β-BODIPY® FL C5-HPA, D3805). We offer several glycerophospholipid analogs labeled with a single greenfluorescent BODIPY® 500/510 or a BODIPY® FL fluorophore or red-orange– fluorescent BODIPY® 530/550 fluorophore on the sn-2 acyl chain, including: • BODIPY® 500/510 dye–labeled glycerophosphocholine (D3793, Figure 13.2.15) • BODIPY® FL dye–labeled glycerophosphocholine (D3792, Figure 13.2.16; D3803, Figure 13.2.17) • BODIPY® 530/550 dye–labeled glycerophosphocholine (D3815, Figure 13.2.18) • BODIPY® FL dye–labeled phosphatidic acid (D3805, Figure 13.2.19) In addition, we prepare a glycerophosphocholine analog with a single nonhydrolyzable ether-linked BODIPY® FL fluorophore on the sn-1 position (D3771, Figure 13.2.20), as well as several doubly labeled glycerophospholipids. These doubly labeled glycerophospholipids, which are discussed in greater detail in Section 17.4, are designed primarily for detection of phospholipase A1 and phospholipase A 2 and include: • Glycerophosphoethanolamine with a BODIPY® FL dye–labeled sn-1 acyl chain and a dinitrophenyl quencher–modified headgroup (PED-A1, A10070; Figure 13.2.21) • Glycerophosphoethanolamine with a BODIPY® FL dye–labeled sn-2 acyl chain and a dinitrophenyl quencher–modified headgroup 96 (PED6, D23739; Figure 13.2.22) • Glycerophosphocholine with two BODIPY® FL dye–labeled acyl chains (bis-BODIPY® FL C11-PC, B7701; Figure 13.2.22) • Glycerophosphocholine with a BODIPY® 558/568 dye–labeled sn-1 alkyl chain and a BODIPY® FL dye–labeled sn-2 acyl chain (Red/Green BODIPY® PC-A2, A10072; Figure 13.2.23) The spectral properties of BODIPY® FL dye–labeled phospholipids are summarized in Table 13.2. Unlike the nitrobenzoxadiazole (NBD) fluorophore, the BODIPY® FL and BODIPY® 500/510 fluorophores are intrinsically lipophilic and readily localize in the membrane’s interior.1 The fluorophore is completely inaccessible to the membrane-impermeant anti–BODIPY® FL antibody (A5770, Section 7.4), which also recognizes the BODIPY® 500/510 derivative. As shown in Figure 13.2.24, the emission spectrum of the BODIPY® 500/510 fluorophore is much narrower than that of the NBD fluorophore. Because both the extinction coefficient of the BODIPY® 500/510 fluorophore and its quantum yield in a lipophilic environment (EC ~90,000 cm–1M–1 and QY ~0.9) are much higher than those of the NBD fluorophore (EC ~20,000 cm–1M–1 and QY ~0.3), much less BODIPY® 500/510 dye–labeled phospholipid is required for labeling membranes.4 H3C N H3C F B N F O (CH2)� C OCH2 CH3(CH2)5 OCH O O CH2O � OCH2CH2NH C OH Figure 13.2.20 2-Decanoyl-1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diazas-indacene-3-propionyl)amino)undecyl)-sn-glycero-3-phosphocholine (D3771). (CH2)5NH Figure 13.2.21 PED-A1 (N-((6-(2,4-DNP)amino)hexanoyl)-1-(BODIPY® FL C5)-2-hexyl-snglycero-3-phosphoethanolamine; A10070). The MolecularProbes® Probes Handbook: Handbook: AA Guide Probesand andLabeling LabelingTechnologies Technologies The Molecular Guide to to Fluorescent Fluorescent Probes ™ 554 O2N IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes NO2 Chapter 13 — Probes for Lipids and Membranes H C 3 Section 13.2 Fatty Acid Analogs and Phospholipids Quenched substrate (bis-BODIPY® FL C11-PC) N F H C 3 H C 3 F B B N N Quenched substrate (PED6) O F (CH ) 2 10 (CH ) 2 10 F O C OCH C OCH H C 3 O + CH O P OCH CH N(CH ) 2 2 2 33 − O O N 2 B N Phospholipase A 2 H C 3 F CH (CH ) 3 2 14 (CH ) F 24 N C OCH O N 2 2 O C OCH O O P OCH CH NH C 2 2 − O CH O 2 (CH ) NH 25 NO 2 Phospholipase A 2 H C 3 H C 3 O N F H C 3 B CH (CH ) 3 2 14 N F (CH ) 2 10 C OCH Fluorescent lysophospholipid CH O 2 2 O + CH O P OCH CH N(CH ) 2 2 2 33 − O F B O O P OCH CH NH C 2 2 − O (CH ) NH 25 NO 2 Nonfluorescent lysophospholipid + N O N 2 2 HOCH HOCH H C 3 C OCH O + (CH ) 2 10 F H C 3 C OH O N F N H C 3 B F (CH ) 24 C OH O N H C 3 Fluorescent fatty acid (BODIPY® FL C11 (D3862)) Fluorescent fatty acid (BODIPY® FL C5 (D3834)) N � F N B F H3C O CH2CH2 C NH(CH2)� OCH2 F N B F (CH2)� C OCH O N O CH2O � OCH2CH2N(CH3)3 O Fluorescence emission Figure 13.2.22 Mechanism of phospholipase activity–linked fluorescence enhancement responses of bis-BODIPY® FL C11-PC (B7701) and PED6 (D23739). Note that enzymatic cleavage of bis-BODIPY® FL C11-PC yields two fluorescent products, whereas cleavage of PED6 yields only one. NBD-PC (N-3787) H3 C Figure 13.2.23 Red/Green BODIPY® PC-A2 (1-O-(6-BODIPY® 558/568-aminohexyl)2-BODIPY® FL C5-sn-glycero-3-phosphocholine; A10072). Figure 13.2.24 Fluorescence spectra (excitation at 475 nm) of β-BODIPY® 500/510 C12-HPC (blue line peak at 516 nm, D3793) and NBD C12-HPC (red line peak at 545 nm, N3787) incorporated in DOPC (dioctadecenoylglycerophosphocholine) liposomes at molar ratios of 1:400 mole:mole (labeled:unlabeled PC). The integrated intensities of the spectra are proportional to the relative fluorescence quantum yields of the two probes. BODIPY®-PC (D-3793) 500 550 600 650 Wavelength (nm) Table 13.2 Spectral properties of some lipid probes. Spectral Property Pyrene DPH NBD BODIPY® FL Ex/Em (nm) * 340/376 360/430 470/530 507/513 QY (τ) † 0.6 (>100 nanoseconds) 0.8 (4–8 nanoseconds) 0.32 (5–10 nanoseconds) 0.9 (6 nanoseconds) Concentration dependence Excimer emission (~470 nm) at high concentrations. Self-quenched at high concentrations. Self-quenched at high concentrations. Excimer emission (~620 nm) at high concentrations. Environmental sensitivity Very sensitive to quenching by oxygen. Essentially nonfluorescent in water. Essentially nonfluorescent in water. Essentially nonfluorescent in water. Relatively insensitive. Strongly fluorescent in both aqueous and lipid environments. * Typical fluorescence excitation and emission maxima for membrane-intercalated probes. † QY = fluorescence quantum yield; τ = fluorescence decay lifetime. Typical values for membraneintercalated probes are listed. These values may show significant environment-dependent variations. ™ The Handbook: A Guide to Fluorescent Probes and Labeling TheMolecular MolecularProbes Probes® Handbook: A Guide to Fluorescent Probes and LabelingTechnologies Technologies IMPORTANT NOTICE: described The products described in this oneLimited or more Use Limited UseLicense(s). Label License(s). Please to the Appendix IMPORTANT NOTICE : The products in this manual are manual coveredare bycovered one or by more Label Please referrefer to the Appendix onon page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes 555 Chapter 13 — Probes for Lipids and Membranes B 1:10 Fluorescence emission Fluorescence emission A 500 600 Section 13.2 Fatty Acid Analogs and Phospholipids 1:5 700 500 Wavelength (nm) 600 700 Wavelength (nm) Figure 13.2.25 A) Fluorescence spectrum of β-C8-BODIPY® 500/510 C5-HPC (D3795) incorporated in DOPC (dioctadecenoylphosphocholine) liposomes at 1:100 mole:mole (labeled:unlabeled PC). B) Fluorescence spectra at high molar incorporation levels: 1:10 mole:mole and 1:5 mole:mole. Incorporation of high molar ratios (>10 mole %) of the BODIPY® 500/510 dye–labeled phospholipids into membranes results in a dramatic spectral shift of the fluorescence emission spectrum to longer wavelengths (Figure 13.2.25). We have also observed this spectral shift in the Golgi of live cells that have been labeled with our BODIPY® dye–labeled ceramides (Section 12.4) and with BODIPY® fatty acids that have been metabolically incorporated by cells (Figure 13.2.2). In fluorescence resonance energy transfer (FRET) measurements, the green-fluorescent BODIPY® 500/510 dye is an excellent donor to longer-wavelength BODIPY® probes 97,98 (Figure 13.2.26) and acceptor from dansyl-, DPH- or pyrene-labeled phospholipids.99 These probe combinations offer several alternatives to the widely used NBD–rhodamine fluorophore pair for researchers using FRET techniques to study lipid transfer and membrane fusion.97 Applications Once cells are labeled with a BODIPY® phospholipid, the probe shows little tendency to spontaneously transfer between cells.100 Consequently, BODIPY® dye–labeled phospholipids have been used in a number of studies of cell membrane structure and properties: Fluorescence emission 1 2 5 3 4 3 4 2 5 500 1 550 600 650 Wavelength (nm) Figure 13.2.26 Fluorescence resonance energy transfer from β-BODIPY® 500/510 C12-HPC (peak at 516 nm, D3793) to BODIPY® 558/568 C12 (peak at 572 nm, D3835) in DOPC (dioctadecenoylglycerophosphocholine) lipid bilayers using 475 nm excitation. Ratio of acceptors to donors is: 1) 0; 2) 0.2; 3) 0.4; 4) 0.8; and 5) 2.0. Figure 13.2.27 Confocal laser-scanning microscopy images of a giant unilamellar phospholipid vesicle (GUV). The lipid composition of this GUV was DPPC/DLPC = 1/1, with DiIC20(3) and β-BODIPY® FL C5-HPC (D3803) dyes at mole fraction ~0.001. Excitation was at 488 nm. The upper left image is the fluorescence emission through a 585 nm longpass filter, thus almost exclusively from DiIC20(3). The lower left image is the emission through a 522 ± 35 nm bandpass filter, thus almost exclusively from β-BODIPY® FL C5-HPC. The right image is color-merged, using the public domain NIH Image program. Image contributed by Gerald W. Feigenson, Cornell University, and reprinted with permission from Biophys J (2001) 80:2775. • Despite their good photostability, BODIPY® lipids are useful for fluorescence recovery after photobleaching (FRAP) measurements of lipid diffusion.101,102 • Researchers have used BODIPY® fatty acids and phospholipids to visualize compartmentalization of specific lipid classes in Schistosoma mansoni 103 and fungi.104,105 • β-BODIPY® FL C12-HPC (D3792) has been used to examine lipid–protein interactions involved in bacterial protein secretion via fluorescence resonance energy transfer (FRET) measurements 106 (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2). • β-BODIPY® FL C5-HPC 107 (D3803) has been used to characterize lipid domains by fluorescence correlation spectroscopy 108 (Fluorescence Correlation Spectroscopy (FCS)—Note 1.3), confocal laser-scanning microscopy 109 (Figure 13.2.27) and near-field scanning optical microscopy.101,110 • bis-BODIPY® FL C11-PC (B7701) has BODIPY® FL dye–labeled sn-1 and sn-2 acyl groups (Figure 13.2.28), resulting in partially quenched fluorescence that increases when one of the acyl groups is hydrolyzed by phospholipase A1 or A 2. The hydrolysis products are BODIPY® FL undecanoic acid (D3862) and BODIPY® FL dye–labeled lysophosphatidylcholine (Figure 13.2.22). The probe has been used successfully in human neutrophils, plants and zebrafish to detect phospholipase A activity 111–116 (Section 17.4). • β-BODIPY® FL C5-HPC (D3803) has been used to investigate the cellular uptake of antineoplastic ether lipids.117 Figure 13.2.28 1,2-bis-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)sn-glycero-3-phosphocholine (bis-BODIPY® FL C11-PC, B7701). TheMolecular MolecularProbes® Probes Handbook: Handbook: AAGuide and Labeling Labeling Technologies Technologies The GuidetotoFluorescent Fluorescent Probes Probes and ™ 556 IMPORTANT NOTICE: The products described in this manual covered one or more Limited Use Label License(s). Please refer to the Appendix IMPORTANT NOTICE : The products described in thisare manual arebycovered by one or more Limited Use Label License(s). Please referonto page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes Chapter 13 — Probes for Lipids and Membranes Section 13.2 Fatty Acid Analogs and Phospholipids Phospholipid with DPH-Labeled Acyl Chain Properties The fluorescent phospholipid analog β-DPH HPC (D476) comprises diphenylhexatriene propionic acid coupled to glycerophosphocholine at the sn-2 position (Figure 13.2.29). It is therefore related to the neutral membrane probe DPH and the cationic derivative TMA-DPH (D202, T204; Section 13.5). DPH and its derivatives exhibit strong fluorescence enhancement when incorporated into membranes, as well as sensitive fluorescence polarization (anisotropy) responses to lipid ordering (Fluorescence Polarization (FP)—Note 1.4). β-DPH HPC was originally devised to improve the localization of DPH in membranes.118,119 Unlike underivatized DPH, it can be used to specifically label one leaflet of a lipid bilayer, facilitating analysis of membrane asymmetry.120 Applications DPH derivatives are predominantly used to investigate the structure and dynamics of the membrane interior either by fluorescence polarization or lifetime measurements. Researchers have used β-DPH HPC as a probe for lipid–protein interactions,121–123 alcohol-induced perturbations of membrane structure,124,125 molecular organization and dynamics of lipid bilayers 11,126–128 and lipid peroxidation.129 Fluorescence lifetime measurements of β-DPH HPC provide a sensitive indicator of membrane fusion.130–132 In addition to membrane fusion, β-DPH HPC has been used to monitor various other lipidtransfer processes.133–135 Applications NBD acyl–modified probes are used for investigating lipid traffic, either by directly visualizing NBD fluorescence,152–155 by exploiting NBD self-quenching 156–158 or by fluorescence resonance energy transfer methods.140,152,159–161 Lateral domains in model monolayers, bilayers and cell membranes have been characterized using NBD phospholipids in conjunction with fluorescence recovery after photobleaching 162–164 (FRAP), fluorescence resonance energy transfer 165 (FRET) (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2) and direct microscopy techniques.166–169 Transmembrane lipid distribution (Lipid-Mixing Assays of Membrane Fusion—Note 13.1) has been assessed using fluorescence resonance energy transfer from NBD HPC to rhodamine DHPE 149,151,170 (L1392) or alternatively by selective dithionite (S2O42–) reduction of NBD phospholipids in the outer membrane monolayer 171 (Figure 13.2.31). Figure 13.2.29 β-DPH HPC (2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero3-phosphocholine; D476). Phospholipids with NBD-Labeled Acyl Chains Properties Our acyl-modified nitrobenzoxadiazole (NBD) phospholipid probes include both the NBD hexanoyl- and NBD dodecanoyl-glycerophosphocholines (NBD C6 -HPC, N3786; Figure 13.2.30 and NBD C12-HPC, N3787). Table 13.2 compares the spectral properties of these probes with those of the BODIPY®, DPH and pyrene lipid probes. Unlike the BODIPY® phospholipids, the location of the relatively polar NBD fluorophore of NBD C6 -HPC and NBD C12-HPC in phospholipid bilayers does not appear to conform to expectations based on the probe structure. A variety of physical evidence indicates that the NBD moiety "loops back" to the head-group region 136–139 (Figure 13.2.1). In fact, the fluorophore in this acyl-modified phospholipid appears to probe the same location as does the head group–labeled glycerophosphoethanolamine derivative NBD-PE 17 (N360). These NBD probes transfer spontaneously between membranes, with NBD C6 -HPC transferring more rapidly than its more lipophilic C12 analog.140,141 NBD C6 -HPC can be readily removed (backexchanged) from the plasma membrane by incubating the labeled cells either with unlabeled lipid vesicles 142 or with bovine serum albumin.143–145 This property is useful for quantitating lipid transfer and for studying phospholipid distribution asymmetry and transmembrane "flip-flop" rates in lipid bilayers.17,146–151 Figure 13.2.30 2-(6-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-snglycero-3-phosphocholine (NBD C6-HPC, N3786). O NH(CH ) 25 N C O OH 2− S O 2 4 NH(CH ) 25 O Fluorescent OH O N NO 2 C N N NH 2 Nonfluorescent Figure 13.2.31 Dithionite reduction of 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid (NBD-X, N316). The elimination of fluorescence associated with this reaction, coupled with the fact that extraneously added dithionite is not membrane permeant, can be used to determine whether the NBD fluorophore is located in the external or internal monolayer of lipid bilayer membranes. ™ The Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies TheMolecular Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes 557 Chapter 13 — Probes for Lipids and Membranes Section 13.2 Fatty Acid Analogs and Phospholipids Phospholipids with Pyrene-Labeled Acyl Chains Properties Figure 13.2.32 1-Hexadecanoyl-2-(1-pyrenedecanoyl)-snglycero-3-phosphocholine (β-py-C10-HPC, H361). Phospholipid analogs with pyrene-labeled sn-2 acyl chains (Figure 13.2.32) are among the most popular fluorescent membrane probes.29,172,173 We offer pyrenedecanoyl-labeled glycerophospholipids with phosphocholine (H361) and phosphoglycerol (H3809) head groups. The spectral properties of the pyrene lipid probes are summarized in Table 13.2. Of primary importance in terms of practical applications is the concentration-dependent formation of excited-state pyrene dimers (excimers), which exhibit a distinctive red-shifted emission (peak ~470 nm) (Figure 13.2.10). Applications Figure 13.2.33 1-Hexadecanoyl-2-(1-pyrenedecanoyl)-snglycero-3-phosphoglycerol, ammonium salt (β-py-C10-PG, H3809). Figure 13.2.34 1,2-Bis-(1-pyrenebutanoyl)-sn-glycero-3phosphocholine (B3781). The excimer-forming properties of pyrene are well suited for monitoring membrane fusion (Lipid-Mixing Assays of Membrane Fusion—Note 13.1) and phospholipid transfer processes.37,173–178 The monomer/excimer emission ratio can also be used to characterize membrane structural domains and their dependence on temperature, lipid composition and other external factors.179–182 Pyrenedecanoyl glycerophosphocholine (β-py-C10-HPC, H361) has been used to elucidate the effect of extrinsic species such as Ca 2+,183 plateletactivating factor,184 drugs,185 membrane-associated proteins 186–188 and ethanol 189 on lipid bilayer structure and dynamics. The anionic phosphoglycerol analog (H3809, Figure 13.2.33) is preferred as a substrate for secretory phosholipases A 2 relative to other phospholipid classes.190,191 The long excited-state lifetime of pyrene (Table 13.2) renders the fluorescence of its conjugates very susceptible to oxygen quenching, and consequently these probes can be used to measure oxygen concentrations in solutions,192 lipid bilayers 193 and cells.194,195 Glycerophospholipids in which both alcohols are esterified to pyrene fatty acids (Figure 13.2.1), as in our bis-(1-pyrenebutanoyl)- and bis-(1-pyrenedecanoyl)glycerophosphocholines (B3781, Figure 13.2.34; B3782) show strong excimer fluorescence, with maximum emission near 470 nm.29 Hydrolysis of either fatty acid ester by a phospholipase results in liberation of a pyrene fatty acid and an emission shift to shorter wavelengths, making these probes useful as phospholipase substrates 196–199 (Section 17.4). NOTE 13.2 Antibodies for Detecting Membrane-Surface Labels For detecting labels at the membrane surface, we offer antibodies that recognize the following labels: • • • • • • • • • • Alexa Fluor® 488 dye (A11094) BODIPY® FL dye (A5770) Alexa Fluor® 405 and Cascade Blue® dyes (A5760) Dansyl (A6398) Dinitrophenyl chromophore (A6423, A6430, A6435, A11097, Q17421MP) Fluorescein and Oregon Green® dyes (A889, A982, A6413, A6421, A11090, A11091, A11095, A11096, Q15421MP, Q15431MP) Green Fluorescent Protein (GFP, A6455, A10259, A10262, A10263, A11120, A11121, A11122, A21311, A21312, A31851, A31852, G10362) Lucifer yellow (A5750, A5751) Tetramethylrhodamine (A6397) Texas Red® dye (A6399) Fluorescent conjugates of several of these anti-dye and anti-hapten antibodies are available; see Section 7.4 and Table 7.8 for complete product information. These antibodies can be used for direct detection of labeled phospholipids via fluorescence quenching1,2 (or fluorescence enhancement, in the case of the anti-dansyl antibody). When used in conjunction with phospholipids with dye-labeled head groups (Table 13.1), they are important tools for: • • • Studies of molecular recognition mechanisms at membrane surfaces3 Lipid diffusion measurements4,5 Quantitation of lipid internalization by endocytosis6,7 In addition to anti-fluorophore antibodies, we offer a selection of streptavidin conjugates (Section 7.6, Table 7.9) for detecting biotinylated phospholipids. 1. Biochemistry (1999) 38:976; 2. J Biol Chem (1998) 273:22950; 3. Angew Chem Int Ed Engl (1990) 29:1269; 4. J Cell Biol (1993) 120:25; 5. Proc Natl Acad Sci U S A (1991) 88:6274; 6. J Cell Biol (1988) 106:1083; 7. Cell (1991) 64:393. The MolecularProbes® Probes Handbook: Handbook: AAGuide Probesand andLabeling LabelingTechnologies Technologies The Molecular Guide to to Fluorescent Fluorescent Probes ™ 558 IMPORTANT NOTICE: The products described in this manual coveredare by covered one or more Limited Use Label License(s). Please refer to thePlease Appendix onto IMPORTANT NOTICE : The products described in thisaremanual by one or more Limited Use Label License(s). refer page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes Chapter 13 — Probes for Lipids and Membranes Section 13.2 Fatty Acid Analogs and Phospholipids Phospholipid with an NBD-Labeled Head Group Phospholipids with a Fluorescent or Biotinylated Head Group Phospholipid with a Dansyl-Labeled Head Group The phospholipid analog incorporating the environment-sensitive 200 dansyl fluorophore (dansyl DHPE, D57; Figure 13.2.35) is a useful probe of lipid–water interfaces.53,201 It is sensitive to the interactions of a number of proteins, including protein kinase C, 202,203 annexins 204,205 and phospholipase A 2,206–208 with membrane surfaces. Dansyl DHPE has also been used to examine the effects of cholesterol on the accessibility of the dansyl hapten to antibodies 209 (Antibodies for Detecting Membrane-Surface Labels—Note 13.2). Phospholipid with a Marina Blue® Dye–Labeled Head Group Marina Blue® DHPE (M12652, Figure 13.2.36) is optimally excited by the intense 365 nm spectral line of the mercury-arc lamp and exhibits bright blue fluorescence emission near 460 nm. Significantly, the pKa value of this 6,8-difluoro-7-hydroxycoumarin derivative is 2–3 log units lower than that of nonfluorinated 7-hydroxycoumarin analogs; consequently, Marina Blue® DHPE is expected to be strongly fluorescent in membranes, even at neutral pH. Phospholipid with a Pacific Blue™ Dye–Labeled Head Group The Pacific Blue™ dye–labeled phospholipid (Pacific Blue™ DMPE, P22652; Figure 13.2.37) is our only head group–labeled phospholipid with tetradecanoyl (myristoyl) esters rather than hexadecanoyl (palmitoyl) esters. This blue-fluorescent phospholipid is structurally similar to a phospholipid described by Gonzalez and Tsien for use in a FRETbased measurement of membrane potential.210 N(CH3)2 (CH3CH2)3NH The widely used membrane probe nitrobenzoxadiazolyldihexadecanoylglycerophosphoethanolamine 17 (NBD-PE, N360; Figure 13.2.38) has three important optical properties: photolability, which makes it suitable for photobleaching recovery measurements; concentration-dependent self-quenching; and fluorescence resonance energy transfer to rhodamine acceptors (usually rhodamine DHPE, L1392). Spectroscopic characteristics of NBD-PE are generally similar to those described for our phospholipids with NBD-labeled acyl chains (N3786, N3787). NBD-PE is frequently used in NBD–rhodamine fluorescence energy transfer experiments to monitor membrane fusion (Lipid-Mixing Assays of Membrane Fusion— Note 13.1). In addition, this method can be used to detect lipid domain formation 165 and intermembrane lipid transfer 211–214 and to determine the transbilayer distribution of phospholipids.151 Attachment of the NBD fluorophore to the head group makes NBD-PE resistant to transfer between vesicles.142 NBD-PE has been used in combination with either rhodamine DHPE (L1392) or Texas Red® DHPE (T1395MP) for visualizing the spatial relationships of lipid populations by fluorescence resonance energy transfer microscopy.215 The nitro group of NBD can be reduced with sodium dithionite, irreversibly eliminating the dye’s fluorescence (Figure 13.2.31). This technique can be employed to determine whether the probe is localized on the outer or inner leaflet of the cell membrane.171,216–218 The argon-ion laser–excitable NBD-PE is also a frequent choice for fluorescence recovery after photobleaching (FRAP) measurements of lateral diffusion in membranes.219–222 In addition, NBD-PE is of particular value for monitoring bilayer-to-hexagonal phase transitions, because these transitions cause an increase in NBD-PE’s fluorescence intensity.223–225 Phospholipid with a Fluorescein-Labeled Head Group Fluorescein-derivatized dihexadecanoylglycerophosphoethanolamine (fluorescein DHPE, F362; Figure 13.2.39) is a membranesurface probe that is sensitive to both the local electrostatic potential and pH.226–228 An anti–fluorescein/Oregon Green® antibody (A889, Section 7.4) has been employed in combination with fluorescein DHPE O CH3(CH2)1� C OCH2 CH3(CH2)1� C OCH O O �O2 CH2O � OCH2CH2NH O Figure 13.2.35 N-(5-dimethylaminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl-sn-glycero3-phosphoethanolamine, triethylammonium salt (dansyl DHPE, D57). Figure 13.2.38 NBD-PE (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero3-phosphoethanolamine, triethylammonium salt; N360). Figure 13.2.36 Marina Blue® 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Marina Blue® DHPE, M12652). (CH3CH2)3NH O CH3(CH2)12 C OCH2 CH3(CH2)12 C OCH O F O O OH O CH2O � OCH2CH2NH C O F O Figure 13.2.37 Pacific Blue™ DMPE (Pacific Blue™ 1,2-ditetradecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt; P22652). Figure 13.2.39 Fluorescein DHPE (N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-snglycero-3-phosphoethanolamine, triethylammonium salt; F362). ™ The Handbook: A Guide to Fluorescent Probes and Labeling Technologies TheMolecular MolecularProbes Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPORTANT NOTICE:described The products described in are this covered manual are by oneLimited or moreUse Limited UseLicense(s). Label License(s). Please to the Appendix IMPORTANT NOTICE : The products in this manual bycovered one or more Label Please referrefer to the Appendix onon page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes 559 Chapter 13 — Probes for Lipids and Membranes Section 13.2 Fatty Acid Analogs and Phospholipids to investigate specific recognition interactions at membrane surfaces 229,230 (Antibodies for Detecting Membrane-Surface Labels—Note 13.2). Because of fluorescein’s photolability, fluorescein DHPE is a useful reagent for measuring lateral diffusion in membranes using fluorescence photobleaching recovery methods.231,232 Another technique, single-particle tracking (SPT), provides direct measurements of diffusion rates by calculating the trajectories of fluorescent polystyrene beads or colloidal gold particles from time-sequential images.233,234 FluoSpheres® fluorescent microspheres (Section 6.5) were labeled with streptavidin and then coupled to fluorescein DHPE using a biotinylated conjugate of anti–fluorescein/Oregon Green® monoclonal 4-4-20 (A6421, Section 7.4). Diffusion rates measured with this bridged conjugate in glass-supported phospholipid bilayers were the same as those determined with streptavidin beads coupled directly to biotin-X DHPE (B1616). Fluorescein DHPE has also been used in conjunction with polyclonal anti–fluorescein/Oregon Green® antibody (A889, Section 7.4) to prepare colloidal gold probes for SPT diffusion measurements in supported phospholipid bilayers and in keratocyte plasma membranes.235 Phospholipid with an Oregon Green® 488 Dye–Labeled Head Group With absorption and emission spectra that are virtually superimposable on those of fluorescein, our Oregon Green® 488 DHPE (O12650, Figure 13.2.40) provides an important alternative to fluorescein DHPE in its many applications. When compared with the fluorescein derivative, Oregon Green® 488 DHPE exhibits greater photostability and a lower pKa (pKa = 4.7 versus 6.4 for fluorescein); however, these pKa values may differ when the probes are bound to membranes. Phospholipid with a BODIPY® FL Dye–Labeled Head Group Our phospholipid with the green-fluorescent BODIPY® FL dye attached to the head group (BODIPY® FL DHPE, D3800; Figure 13.2.41) has significant potential for studies of molecular recognition interactions at membrane surfaces (Antibodies for Detecting MembraneSurface Labels—Note 13.2). Spectral properties of this BODIPY® probe is generally the same as those described above for phospholipids with BODIPY® FL dye–labeled acyl chains. Phospholipids with a Rhodamine or Texas Red® Dye–Labeled Head Group The rhodamine-labeled phospholipids TRITC DHPE (T1391, Figure 13.2.42) and rhodamine DHPE (often referred to as N-Rh-PE, L1392; Figure 13.2.1) do not readily transfer between separated lipid bilayers.140,236 This property has led to the extensive use of rhodamine DHPE for membrane fusion assays based on fluorescence resonance energy transfer from NBD-PE (Lipid-Mixing Assays of Membrane Fusion— Note 13.1). In addition, these probes are good resonance energy transfer acceptors from fluorescent lipid analogs such as the BODIPY® and NBD phospholipids 237 and from protein labels such as 5-iodoacetamidofluorescein 5-IAF, I30451; Section 2.2) and IAEDANS 238,239 (I14, Section 2.3). Rhodamine-labeled phospholipids have also been used as tracers for membrane traffic during endocytosis 240 and for lipid processing in hepatocytes.241 Texas Red® DHPE (T1395MP) is principally employed as an energy transfer acceptor from NBD, BODIPY® and fluorescein lipid probes. The longer emission wavelength of the Texas Red® dye provides superior separation of the donor and acceptor emission signals in resonance energy transfer microscopy.216,242 This technique has enabled visualization of ATP-dependent fusion of liposomes with the Golgi apparatus.243 Membrane flux during hemagglutinin-mediated cell–cell fusion has been visualized using Texas Red® DHPE and the lipophilic carbocyanine DiI (D282, D3911; Section 13.4) as membrane labels.244 Phospholipids with a Biotinylated Head Group We offer phospholipids labeled with a biotin at the head group to facilitate binding of labeled membranes to other biomolecules. The biotinylated phospholipids (biotin DHPE, B1550; biotin-X DHPE, B1616, Figure 13.2.43) can be used to couple avidin or streptavidin (Table 7.9) to cell membranes, liposomes and lipid monolayers.245–248 Avidin can then be employed as a bridge for antibody coupling or for assembling liposomes into multiplex structures.249,250 Liposomes incorporating biotinylated phospholipids can also be used to immobilize membranebound proteins for analysis by affinity chromatography.251 Interactions of biotinylated lipids with streptavidin provide a model for molecular (CH3)2N O (CH3CH2)3NH CH3(CH2)�� C OCH2 CH3(CH2)�� C OCH O N(CH3)2 C O O CH2O � OCH2CH2NH C NH O O O � Figure 13.2.42 TRITC DHPE (N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt; T1391). Figure 13.2.40 Oregon Green® 488 DHPE (Oregon Green® 488 1,2-dihexadecanoyl-sn-glycero3-phosphoethanolamine; O12650). (CH3CH2)3NH O CH3(CH2)�� C OCH2 CH3(CH2)�� C OCH O CH3 N O CH2O � OCH2CH2NH C CH2CH2 O F B N F CH3 O Figure 13.2.41 N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (BODIPY® FL DHPE, D3800). Figure 13.2.43 Biotin-X DHPE (N-((6-(biotinoyl)amino)hexanoyl)-1,2-dihexadecanoyl-snglycero-3-phosphoethanolamine, triethylammonium salt; B1616). The MolecularProbes® Probes Handbook: Handbook: AAGuide and Labeling LabelingTechnologies Technologies The Molecular Guideto toFluorescent Fluorescent Probes Probes and ™ 560 IMPORTANT NOTICE: The products described in this manual covered bycovered one or more Limited Use Label License(s). Please refer to thePlease Appendix IMPORTANT NOTICE : The products described in thisaremanual are by one or more Limited Use Label License(s). referonto page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes Chapter 13 — Probes for Lipids and Membranes Section 13.2 Fatty Acid Analogs and Phospholipids recognition processes at membrane surfaces.252–254 The phase structure of lipid assemblies incorporating biotinylated phospholipids has been studied by X-ray diffraction,218 31P NMR and differential scanning calorimetry.255,256 LipidTOX™ Phospholipid and Neutral Lipid Stains for High-Content Screening With the resolution inherent in an image-based methodology and the productivity of high-throughput assays, high-content screening (HCS) or automated imaging provides a powerful tool for studying biology in a spatial and temporal context. Using HCS technology, researchers can examine multiple cellular targets and parameters in a large number of individually imaged cells and quantitatively assess the data. While many Molecular Probes® products can be directly applied to HCS protocols, we have developed validated tools and assays specifically for HCS platforms. These HCS products are: • Validated on multiple imaging platforms • Packaged in automation-compatible formulations • Compatible with multiplex applications Although designed for HCS platforms, HCS products and kits can also be used with conventional fluorescence microscopes equipped with standard optical filter sets. HCS LipidTOX™ Phospholipidosis Detection Reagents Phospholipidosis is often triggered by cationic amphiphilic drugs, which become enriched in lysosomes to high concentrations and inhibit normal metabolism of phospholipids. The subsequent intracellular accumulation of phospholipids and formation of lamellar bodies— phospholipidosis—can be detected in cells incubated in the presence of phospholipids conjugated to fluorescent dyes. HCS LipidTOX™ Green and HCS LipidTOX™ Red phospholipidosis detection reagents (H34350, H34351), also called LipidTOX™ phospholipid stains, were specifically developed to characterize the potentially toxic side effects of compounds on lipid metabolism in mammalian cell lines using image-based HCS assays.257 Key advantages of this series of phospholipidosis detection reagents over conventional stains such as NBD-PE (N360) include their ready-to-use aqueous formulation, their narrow emission profiles (excitation/emission maxima ~495/525 nm for HCS LipidTOX™ Green phospholipidosis detection reagent and ~595/615 nm for HCS LipidTOX™ Red phospholipidosis detection reagent) and their compatibility with HCS LipidTOX™ neutral lipid stains. HCS LipidTOX™ phospholipidosis detection reagents have not been observed to affect the normal growth of cells, and their live-cell staining patterns are maintained after formaldehyde fixation. These reagents are designed for fixed–end point workflows in which formaldehyde-fixed cells in microplates are processed, imaged and analyzed. HCS LipidTOX™ phospholipidosis detection reagents can easily be detected with fluorescence microscopes or HCS readers equipped with standard filter sets. HCS LipidTOX™ Neutral Lipid Stains As with phospholipidosis, steatosis or the intracellular accumulation of neutral lipids as lipid droplets or globules is often triggered by drugs that affect the metabolism of fatty acids or neutral lipids. HCS LipidTOX™ neutral lipid stains were developed to characterize the effects of drugs and other compounds on lipid metabolism in mammalian cell lines. HCS LipidTOX™ neutral lipid stains have an extremely high affinity for neutral lipid droplets. These reagents are added after cell fixation and do not require subsequent wash steps after incubation with the sample. Key advantages of this series of neutral lipid stains over conventional stains such as nile red (N1142; Section 13.5) include their ready-to-use formulations, their flexibility for multiplexing protocols and their compatibility with HCS LipidTOX™ phospholipidosis detection reagents. HCS LipidTOX™ neutral lipid stains can also be used to monitor the formation and differentiation of adipocytes, a process called adipogenesis. Adipogenesis is of acute interest to the biomedical and drug discovery community as it plays an important role in diseases such as obesity, diabetes and atherosclerosis. Described more thoroughly in Section 13.5, HCS LipidTOX™ neutral lipid stains are available with green, red and deep red fluorescence emission: • HCS LipidTOX™ Green neutral lipid stain (H34475), with excitation/emission maxima ~495/505 nm (Figure 13.2.44) • HCS LipidTOX™ Red neutral lipid stain (H34476), with excitation/ emission maxima ~577/609 nm • HCS LipidTOX™ Deep Red neutral lipid stain (H34477), with excitation/emission maxima ~637/655 nm HCS LipidTOX™ neutral lipid stains are designed for fixed–end point workflows in which formaldehyde-fixed cells in microplates are processed, imaged and analyzed. These stains can easily be detected with fluorescence microscopes or HCS readers equipped with standard filter sets. Figure 13.2.44 LipidTOX™ Green neutral lipid stain and fatty acid–binding protein (FABP4) antibody labeling in adipocytes. Adipocytes differentiated from 3T3-L1 mouse fibroblasts were fixed with formaldehyde and permeabilized with saponin before labeling with rabbit anti–fatty acid binding protein (FABP4) IgG (red). These cells were then stained with LipidTOX™ Green neutral lipid stain (H34475, green), counterstained with DAPI (D1306, D21490; blue) and mounted in ProLong® Gold antifade reagent (P36930). ™ The Handbook: A Guide to Fluorescent Probes and Labeling Technologies TheMolecular MolecularProbes Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPORTANT NOTICE: described The products described in are this manual by oneLimited or moreUse Limited UseLicense(s). Label License(s). Please to the Appendix IMPORTANT NOTICE : The products in this manual coveredare bycovered one or more Label Please referrefer to the Appendix onon page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes 561 Chapter 13 — Probes for Lipids and Membranes Section 13.2 Fatty Acid Analogs and Phospholipids HCS LipidTOX™ Phospholipidosis and Steatosis Detection Kit The detection and analysis of prelethal mechanisms in toxicological profiling and compound screening are extremely important components of the drug discovery process. The cationic amphiphilic drugs are among the most prominent examples of compounds that impact lipid metabolism of cells. These drugs tend to become enriched in lysosomes to high concentrations and inhibit the normal metabolism of phospholipids, which in turn causes the intracellular accumulation of phospholipids and the formation of lamellar bodies. Other drug classes more adversely affect various aspects of fatty acid or neutral lipid metabolism, leading to the cytoplasmic accumulation of neutral lipid as lipid droplets or globules. The HCS LipidTOX™ Phospholipidosis and Steatosis Detection Kit (H34157, H34158) provides a complete set of reagents for performing validated HCS assays to detect and distinguish these two facets of cytotoxicity—the intracellular accumulation of phospholipids (phospholipidosis) and of neutral lipids (steatosis)—in mammalian cell lines after exposure to test compounds.258 This kit includes an aqueous, red-fluorescent formulation of labeled phospholipids (LipidTOX™ Red phospholipid stain, excitation/emission ~595/615 nm) and a ready-to-use, highly selective green-fluorescent stain for neutral lipids (LipidTOX™ Green neutral lipid stain, excitation/emission ~495/505 nm), which can be used sequentially for the analysis of phospholipidosis and steatosis, respectively, or can be used separately for single-parameter analysis. After incubation with LipidTOX™ Red phospholipid stain and a test compound, the cells are fixed with formaldehyde and labeled with LipidTOX™ Green neutral lipid stain (Figure 13.2.45). Neither LipidTOX™ Red phospholipid stain, nor LipidTOX™ Green phospholipid stain described above, requires sonication or organic solvents. Furthermore, LipidTOX™ Green neutral lipid stain (as well as the other LipidTOX™ neutral lipid stains described above) is more selective than nile red, allowing you to easily distinguish neutral lipids (such as those in adipocytes and cells undergoing steatosis) from other types of lipids. Figure 13.2.45 Multiplex detection of phospholipidosis and steatosis in HepG2 cells using the HCS LipidTOX™ Phospholipidosis and Steatosis Detection Kit (H34157, H34158). HepG2 cells were co-incubated with tamoxifen and LipidTOX™ Red phospholipid stain, followed by fixation with formaldehyde and labeling with HCS LipidTOX™ Green neutral lipid stain and Hoechst 33342 (H1399, H3570, H21492). Each HCS LipidTOX™ Phospholipidosis and Steatosis Detection Kit provides: • • • • LipidTOX™ Red phospholipid stain LipidTOX™ Green neutral lipid stain Hoechst 33342 for nuclear labeling Propranolol, a positive-control compound for inducing phospholipidosis • Cyclosporin A, a positive-control compound for inducing steatosis • Dimethylsulfoxide (DMSO) • Detailed protocols Sufficient reagents are provided for 240 assays (H34157, 2-plate size) or 1200 assays (H34158, 10-plate size), based on assay volumes of 100 µL per well. These kits are designed for fixed–end point workflows in which formaldehyde-fixed cells in microplates are processed, imaged and analyzed. The fluorescent stains used for the analysis of phospholipidosis and steatosis can easily be detected with fluorescence microscopes or HCS readers equipped with standard filter sets. REFERENCES 1. Biochim Biophys Acta (1998) 1375:13; 2. Biochemistry (1992) 31:5312; 3. Chem Phys Lipids (2002) 116:3; 4. Anal Biochem (1991) 198:228; 5. Biochem Biophys Res Commun (1992) 187:1594; 6. J Biol Chem (2001) 276:1391; 7. J Biol Chem (1997) 272:8531; 8. Exp Parasitol (1997) 86:133; 9. Am J Physiol (1995) 269:G842; 10. Methods Enzymol (2000) 319:603; 11. Biochim Biophys Acta (2000) 1487:61; 12. Anal Biochem (1998) 265:290; 13. FEBS Lett (1999) 453:278; 14. Free Radic Biol Med (2002) 33:473; 15. J Biochem Biophys Methods (1997) 35:23; 16. Photochem Photobiol (1991) 54:361; 17. Chem Phys Lipids (1990) 53:1; 18. Biochemistry (1995) 34:11919; 19. Chem Phys Lipids (1991) 58:111; 20. Biophys J (2001) 80:832; 21. J Am Chem Soc (1975) 97:4114; 22. Anal Biochem (1988) 174:101; 23. 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J Cell Biol (1991) 115:245; 223. Biophys J (1992) 63:309; 224. Biochemistry (1990) 29:2976; 225. Biochemistry (1988) 27:3947; 226. Z Naturforsch C (2000) 55:418; 227. Biochim Biophys Acta (1998) 1374:63; 228. J Biol Chem (1999) 274:29951; 229. Biophys J (1992) 63:823; 230. J Am Chem Soc (1981) 103:6797; 231. Biophys J (1994) 66:25; 232. J Cell Biol (1986) 103:807; 233. J Membr Biol (1993) 135:83; 234. Proc Natl Acad Sci U S A (1991) 88:6274; 235. J Cell Biol (1993) 120:25; 236. Biochemistry (1985) 24:6390; 237. Biochemistry (1981) 20:4093; 238. J Biol Chem (1991) 266:12082; 239. Biochemistry (1990) 29:1607; 240. Eur J Cell Biol (1990) 53:173; 241. Biochem J (1992) 284:259; 242. J Cell Biol (1986) 103:1221; 243. Cell (1988) 55:797; 244. J Cell Biol (1993) 121:543; 245. J Immunol Methods (1993) 158:183; 246. Anal Biochem (1992) 207:341; 247. Biophys J (1991) 59:387; 248. Biochim Biophys Acta (1990) 1028:73; 249. Anal Chem (2001) 73:91; 250. Science (1994) 264:1753; 251. Anal Biochem (2000) 280:94; 252. Angew Chem Int Ed Engl (1990) 29:1269; 253. Anal Biochem (1994) 217:128; 254. Biophys J (1993) 65:2160; 255. Biophys J (1994) 66:31; 256. Biochemistry (1993) 32:9960; 257. Toxicol Sci (2007) 99:162; 258. Cytometry A (2009) 77:231. DATA TABLE 13.2 FATTY ACID ANALOGS AND PHOSPHOLIPIDS Cat. No. A3880 A10070 A10072 B1550 B1616 B3781 B3782 B3824 B7701 D57 D94 D476 D3771 D3792 D3793 D3800 D3803 D3805 D3815 D3821 D3822 D3823 D3825 D3832 D3834 MW ~15,350 880.68 986.67 1019.45 1132.61 797.88 966.20 404.31 1029.80 1026.44 434.59 782.01 854.86 895.95 881.93 1067.23 797.77 746.68 921.91 474.44 418.33 404.31 404.31 542.47 320.15 Storage FF,L,AA FF,D,L FF,D,L FF,D FF,D FF,D,L FF,D,L F,L FF,D,L FF,D,L F,L FF,D,L FF,D,L FF,D,L FF,D,L FF,D,L FF,D,L FF,D,L FF,D,L F,L F,L F,L F,L F,L F,L Soluble H2O DMSO DMSO see Notes see Notes see Notes see Notes DMSO see Notes see Notes DMSO, EtOH see Notes see Notes see Notes see Notes see Notes see Notes see Notes see Notes DMSO DMSO DMSO DMSO DMSO DMSO, MeCN Abs 365 505 505 <300 <300 342 340 509 505 336 335 354 506 506 509 505 503 504 534 505 505 508 509 534 505 EC 10,500 92,000 85,000 75,000 62,000 101,000 123,000 4500 4800 81,000 71,000 86,000 86,000 87,000 80,000 79,000 64,000 90,000 87,000 97,000 100,000 76,000 96,000 Em 432 512 567 none none 471 473 515 512 517 519 428 512 513 513 511 512 511 552 512 511 514 515 552 511 Solvent H2O MeOH MeOH EtOH EtOH MeOH MeOH MeOH MeOH MeOH EtOH EtOH EtOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH Notes 1 16 17, 18 2 2 3 4 5 2, 6 2 2, 7 2 2, 5 2, 5 2, 5 2, 5 2, 5 2, 5 5 5 5 5 5 5 continued on next page ™ The Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies TheMolecular Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes 563 Chapter 13 — Probes for Lipids and Membranes Section 13.2 Fatty Acid Analogs and Phospholipids DATA TABLE 13.2 FATTY ACID ANALOGS AND PHOSPHOLIPIDS—continued Cat. No. MW Storage Soluble Abs EC Em Solvent Notes D3835 472.40 F,L DMSO 559 91,000 568 MeOH 5 D3861 504.43 F,L DMSO 582 140,000 591 MeOH 8 D3862 404.31 F,L DMSO 505 92,000 510 MeOH 5 D23739 1136.13 FF,D,L DMSO 505 92,000 511 MeOH 2, 9 F362 1182.54 FF,D,L see Notes 496 88,000 519 MeOH 2, 10 H361 850.13 FF,D,L see Notes 342 37,000 376 MeOH 2, 11, 12 H3809 856.09 FF,D,L see Notes 341 38,000 376 MeOH 2, 11, 12 495 84,000 525 MeOH 15 H34350 ~1100 F,L H2O 595 112,000 615 MeOH 15 H34351 ~1400 F,L H2O L1392 1333.81 FF,D,L see Notes 560 75,000 581 MeOH 2 M12652 944.14 FF,D,L see Notes 365 18,000 460 MeOH 2, 10 N316 294.27 L DMSO 467 23,000 539 MeOH 13 N360 956.25 FF,D,L see Notes 463 21,000 536 MeOH 2, 13 N678 378.43 L DMSO 467 24,000 536 MeOH 13 N3786 771.89 FF,D,L see Notes 465 21,000 533 EtOH 2, 13 N3787 856.05 FF,D,L see Notes 465 22,000 534 EtOH 2, 13 O12650 1086.25 FF,D,L see Notes 501 85,000 526 MeOH 2, 10 P31 372.51 L DMF, DMSO 341 43,000 377 MeOH 11, 12 P96 400.56 L DMF, DMSO 341 44,000 377 MeOH 11, 12 P243 456.67 L DMF, DMSO 341 43,000 377 MeOH 11, 12 P1903MP 288.35 L DMF, DMSO 341 43,000 376 MeOH 11, 12 P22652 961.17 FF,D,L see Notes 411 40,000 454 MeOH 2 P36005 276.42 FF,LL,AA EtOH 304 77,000 416 MeOH 14, 15 T1391 1236.68 FF,D,L see Notes 540 93,000 566 MeOH 2 T1395MP 1381.85 FF,D,L see Notes 583 115,000 601 MeOH 2 For definitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages. Notes 1. ADIFAB fatty acid indicator is a protein conjugate with a molecular weight of approximately 15,350. Em shifts from about 432 nm to 505 nm upon binding of fatty acids. (Mol Cell Biochem (1999) 192:87) 2. Chloroform is the most generally useful solvent for preparing stock solutions of phospholipids (including sphingomyelins). Glycerophosphocholines are usually freely soluble in ethanol. Most other glycerophospholipids (phosphoethanolamines, phosphatidic acids and phosphoglycerols) are less soluble in ethanol, but solutions up to 1–2 mg/mL should be obtainable, using sonication to aid dispersion if necessary. Labeling of cells with fluorescent phospholipids can be enhanced by addition of cyclodextrins during incubation. (J Biol Chem (1999) 274:35359) 3. Phospholipase A cleavage generates a fluorescent fatty acid (P1903MP) and a fluorescent lysophospholipid. 4. Phospholipase A cleavage generates a fluorescent fatty acid (P31) and a fluorescent lysophospholipid. 5. The absorption and fluorescence spectra of BODIPY® derivatives are relatively insensitive to the solvent. 6. Phospholipase A cleavage results in increased fluorescence with essentially no wavelength shift. The cleavage products are D3862 and a fluorescent lysophospholipid. 7. Diphenylhexatriene (DPH) and its derivatives are essentially nonfluorescent in water. Absorption and emission spectra have multiple peaks. The wavelength, resolution and relative intensity of these peaks are environment dependent. Abs and Em values are for the most intense peak in the solvent specified. 8. Oxidation of the polyunsaturated butadienyl portion of the BODIPY® 581/591 dye results in a shift of the fluorescence emission peak from ~590 nm to ~510 nm. (Methods Enzymol (2000) 319:603, FEBS Lett (1999) 453:278) 9. Phospholipase A2 cleavage results in increased fluorescence with essentially no wavelength shift. The cleavage products are D3834 and a dinitrophenylated lysophospholipid. 10. Spectra of this compound are in methanol containing a trace of KOH. 11. Alkylpyrene fluorescence lifetimes are up to 110 nanoseconds and are very sensitive to oxygen. 12. Pyrene derivatives exhibit structured spectra. The absorption maximum is usually about 340 nm with a subsidiary peak at about 325 nm. There are also strong absorption peaks below 300 nm. The emission maximum is usually about 376 nm with a subsidiary peak at 396 nm. Excimer emission at about 470 nm may be observed at high concentrations. 13. Fluorescence of NBD and its derivatives in water is relatively weak. QY and τ increase and Em decreases in aprotic solvents and other nonpolar environments relative to water. (Biochemistry (1977) 16:5150, Photochem Photobiol (1991) 54:361) 14. Cis-parinaric acid is highly oxygen sensitive. Use under N2 or Ar. Cis-parinaric acid is essentially nonfluorescent in water. 15. This product is supplied as a ready-made solution in the solvent indicated under "Soluble." 16. Phospholipase A1 cleavage results in increased fluorescence with essentially no wavelength shift. The cleavage products are D3834 and a dinitrophenylated lysophospholipid. 17. A10072 exhibits dual emission (Em = 510 nm and 567 nm in MeOH, 513 nm and 575 nm when incorporated in phospholipid bilayer membranes). Phospholipase A2 cleavage results in increased 510–513 nm emission and reciprocally diminshed 567–575 nm emission. 18. A10072 is also soluble at 2 mM in 2-methoxyethanol. The MolecularProbes® Probes Handbook: Handbook: AA Guide Probesand andLabeling LabelingTechnologies Technologies The Molecular Guide to to Fluorescent Fluorescent Probes ™ 564 IMPORTANT NOTICE: The products described in this manual coveredare by covered one or more Limited Use Label License(s). Please refer to thePlease Appendix onto IMPORTANT NOTICE : The products described in thisaremanual by one or more Limited Use Label License(s). refer page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes Chapter 13 — Probes for Lipids and Membranes Section 13.2 Fatty Acid Analogs and Phospholipids PRODUCT LIST 13.2 FATTY ACID ANALOGS AND PHOSPHOLIPIDS Quantity Cat. No. Product A3880 ADIFAB fatty acid indicator 200 µg B1550 biotin DHPE (N-(biotinoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt) 10 mg B1616 biotin-X DHPE (N-((6-(biotinoyl)amino)hexanoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt) B7701 1,2-bis-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine (bis-BODIPY® FL C11-PC) 5 mg 100 µg B3781 1,2-bis-(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine 1 mg B3782 1,2-bis-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine 1 mg B3824 BODIPY® 500/510 C4, C9 (5-butyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-nonanoic acid) 1 mg D3771 2-decanoyl-1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)undecyl)-sn-glycero-3-phosphocholine 1 mg D3822 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (BODIPY® FL C12) D3792 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (β-BODIPY® FL C12-HPC) D3821 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid (BODIPY® FL C16) 1 mg D3834 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid (BODIPY® FL C5) 1 mg 1 mg 100 µg D3805 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphate, diammonium salt (β-BODIPY® FL C5-HPA) 100 µg D3803 D3800 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (β-BODIPY® FL C5-HPC) N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (BODIPY® FL DHPE) 100 µg D3862 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BODIPY® FL C11) 1 mg D3832 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (BODIPY® 530/550 C12) 1 mg 100 µg D3815 2-(4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (β-BODIPY® 530/550 C5-HPC) D3823 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (BODIPY® 500/510 C1, C12) D3793 2-(4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (β-BODIPY® 500/510 C12-HPC) D3825 4,4-difluoro-5-octyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid (BODIPY® 500/510 C8, C5) 1 mg D3861 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BODIPY® 581/591 C11) 1 mg D3835 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (BODIPY® 558/568 C12) D94 11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid (DAUDA) D57 D23739 N-(5-dimethylaminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (dansyl DHPE) N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3phosphoethanolamine, triethylammonium salt (PED6) D476 β-DPH HPC (2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine) 1 mg 100 µg 1 mg 100 µg 1 mg 100 mg 25 mg 1 mg F362 fluorescein DHPE (N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt) 5 mg H34350 HCS LipidTOX™ Green phospholipidosis detection reagent *1000X aqueous solution* *for cellular imaging* *10-plate size* each H34157 HCS LipidTOX™ Phospholipidosis and Steatosis Detection Kit *for high-content screening* *for cellular imaging* *2-plate size* 1 kit H34158 HCS LipidTOX™ Phospholipidosis and Steatosis Detection Kit *for high-content screening* *for cellular imaging* *10-plate size* 1 kit H34351 HCS LipidTOX™ Red phospholipidosis detection reagent *1000X aqueous solution* *for cellular imaging* *10-plate size* each H361 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (β-py-C10-HPC) 1 mg H3809 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoglycerol, ammonium salt (β-py-C10-PG) 1 mg L1392 Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (rhodamine DHPE) 5 mg M12652 Marina Blue® 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Marina Blue® DHPE) 1 mg N360 NBD-PE (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt) N316 NBD-X (6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid) 100 mg 10 mg 100 mg N678 12-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoic acid N3787 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD C12-HPC) 5 mg N3786 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD C6-HPC) 5 mg O12650 Oregon Green® 488 DHPE (Oregon Green® 488 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine) 1 mg P22652 Pacific Blue™ DMPE (Pacific Blue™ 1,2-ditetradecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt) 1 mg P36005 cis-parinaric acid *3 mM in ethanol* A10070 PED-A1 (N-((6-(2,4-DNP)amino)hexanoyl)-1-(BODIPY® FL C5)-2-hexyl-sn-glycero-3-phosphoethanolamine) *phospholipase A1 selective substrate* 100 µg 100 mg 10 mL P1903MP 1-pyrenebutanoic acid *high purity* P31 1-pyrenedecanoic acid 25 mg P96 1-pyrenedodecanoic acid 25 mg P243 1-pyrenehexadecanoic acid A10072 Red/Green BODIPY® PC-A2 (1-O-(6-BODIPY® 558/568-aminohexyl)-2-BODIPY® FL C5-sn-glycero-3-phosphocholine) *ratiometric phospholipase A2 substrate* 5 mg 100 µg T1395MP Texas Red® DHPE (Texas Red® 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt) 1 mg T1391 TRITC DHPE (N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt) 1 mg ™ The Handbook: A Guide to Fluorescent Probes and TheMolecular MolecularProbes Probes® Handbook: A Guide to Fluorescent Probes andLabeling LabelingTechnologies Technologies IMPORTANT NOTICE: described The products described in this oneLimited or more Limited UseLicense(s). Label License(s). Please to the Appendix IMPORTANT NOTICE : The products in this manual are manual coveredarebycovered one or by more Use Label Please referrefer to the Appendix onon page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes 565 Chapter 13 — Probes for Lipids and Membranes Section 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes Sphingolipids Structure and Activity Sphingolipids are essential components of the plasma membrane of eukaryotic cells, where they are typically found in the outer leaflet. Although particularly abundant in mammalian cells, sphingolipids are also present in Saccharomyces cerevisiae,1 other fungi and plants. Sphingolipids differ from phospholipids in that they are based on a lipophilic amino alcohol (sphingosine, Figure 13.3.1) rather than glycerol. Sphingolipids play important roles in signal transduction processes2,3 (Chapter 17). Genetic defects in enzymes in the metabolic pathways of sphingolipid synthesis and degradation, including those involved in type I Gaucher (Ashkenazi) disease, type A Niemann– Pick disease, Krabbe disease,4–8 and other lysosomal storage diseases, can be detected at the cellular level using our fluorescent analogs of sphingolipids. Ceramides are the biological building blocks of more complex sphingolipids. Metabolism of ceramides typically occurs in Golgi and endoplasmic reticulum membranes, and fluorescent ceramide analogs (Section 12.4) are important probes for measuring the intracellular distribution and transport of the labeled molecules in live cells.9 If the hydroxyl group of the ceramide is esterified to phosphocholine, the sphingolipid is a sphingomyelin (Figure 13.3.1). The main pathway of sphingomyelin biosynthesis in mammalian cells is based on the transfer of phosphocholine from glycerophosphocholine to ceramide, catalyzed by sphingomyelin synthase in the Golgi membrane. Synthesis is followed by exocytosis of the sphingomyelin to the plasma membrane, apparently via a vesicular pathway and flip-flop to the outer membrane.2 Sphingomyelinases, which are functionally analogous to phospholipase C in their chemistry, hydrolyze sphingomyelins back to ceramides. Generation of ceramides by hydrolysis of sphingomyelins appears to play a role in mediating the effects of exposure to tumor necrosis factor–α10 (TNF-α), γ-interferon and several other agents, all of which induce an apoptosis-like cell death.11–15 Section15.5 describes our extensive selection of reagents for following the diverse morphological HOCH + (CH ) NCH CH O 33 2 2 2 CH CH NH OH 2 O Figure 13.3.2 BODIPY® FL C12-glucocerebroside. Figure 13.3.3 BODIPY® FL C5-ganglioside GM1 (B13950). CH(CH ) CH 2 12 3 Sphingosine O P CH and biochemical changes that occur during apoptosis. Sensitive fluorescence-based measurements of sphingomyelinase activity using natural, unlabeled sphingomyelin as the substrate can be carried out using our Amplex® Red Sphingomyelinase Assay Kit (A12220), described below. In glycosylsphingolipids, the free hydroxyl group of the ceramide is glycosylated to give a sphingosyl glycoside (cerebroside, Figure 13.3.2) or a ganglioside (Figure 13.3.3). These glycosphingolipids form cell-type–specific patterns at the cell surface that change with cell growth, differentiation, viral transformation and oncogenesis.16 − O CH 2 CH CH NH OH C CH HO CH(CH ) CH 2 12 3 OH O O O CH 2 HO R HOCH 2 CH CH NH OH C CH CH NH OH C OH Sphingomyelin CH CH(CH ) CH 2 12 3 CH CH(CH ) CH 2 12 3 O R Cerebroside O R Ceramide Figure 13.3.1 Sphingomyelins, ceramides and cerebrosides are examples of sphingolipids derived from sphingosine. R represents the hydrocarbon tail portion of a fatty acid residue. TheMolecular MolecularProbes® Probes Handbook: Handbook: AAGuide and Labeling LabelingTechnologies Technologies The Guideto toFluorescent Fluorescent Probes Probes and ™ 566 IMPORTANT NOTICE: The products described in this manual covered bycovered one or more Limited Use Label License(s). Please refer to thePlease Appendix IMPORTANT NOTICE : The products described in thisare manual are by one or more Limited Use Label License(s). referonto page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes Chapter 13 — Probes for Lipids and Membranes Section 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes Glycosphingolipids interact at the cell surface with toxins, viruses and bacteria, as well as with receptors and enzymes17 and are involved in cell-type–specific adhesion processes.16 Gangliosides modulate the trophic factor–stimulated dimerization, tyrosine phosphorylation and subsequent signal transduction events of several tyrosine kinase receptors.17 Ganglioside GM1 has anti-neurotoxic, neuroprotective and neurorestorative effects on various central neurotransmitter systems.18 Gangliosides, including ganglioside GM1, partition into lipid rafts—detergent-insoluble, sphingolipid- and cholesterol-rich membrane microdomains that form lateral assemblies in the plasma membrane.19–25 We offer Vybrant® Lipid Raft Labeling Kits (V34403, V34404, V34405), as well as Alexa Fluor® dye conjugates of subunit B of cholera toxin (Section 7.7), a protein that selectively binds to ganglioside GM1 in lipid rafts. Figure 13.3.4 N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-s-indacene-3-pentanoyl)sphingosine (BODIPY® FL C5-ceramide, D3521). BODIPY® Sphingolipids Ceramides (N-acylsphingosines), like diacylglycerols, are lipid second messengers that function in signal transduction processes.26–28 The concentration-dependent spectral properties of BODIPY® FL C5-ceramide (D3521, B22650; Figure 13.3.4), BODIPY® FL C5-sphingomyelin29–31 (D3522, Figure 13.3.5) and BODIPY® FL C12-sphingomyelin32 (D7711) make them particularly suitable for investigating sphingolipid transport, metabolism and microdomains, 31,33–37 in addition to their well-documented use as structural markers for the Golgi complex 38 (Section 12.4, Figure 13.3.6). BODIPY® FL C5-ceramide can be visualized by fluorescence microscopy39,40 (Figure 13.3.7, Figure 13.3.8) or by electron microscopy following diaminobenzidine (DAB) photoconversion to an electron-dense product41 (Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents—Note 14.2). Our range of BODIPY® sphingolipids also includes the long-wavelength light–excitable BODIPY® TR ceramide42,43 (D7540, Figure 13.3.9), as well as BODIPY® FL C5-lactosylceramide44–49 (D13951), BODIPY® FL C5-ganglioside GM150 (B13950, Figure 13.3.3) and BODIPY® FL C12galactocerebroside51 (D7519). All Molecular Probes® sphingolipids are prepared from D-erythrosphingosine and therefore have the same stereochemical conformation as natural biologically active sphingolipids.52 Complexing fluorescent lipids with bovine serum albumin (BSA) facilitates cell labeling by eliminating the need for organic solvents to dissolve the lipophilic probe—the BSA-complexed probe can be directly dissolved in water. We offer four BODIPY® sphingolipid–BSA complexes for the study of lipid metabolism and trafficking, including BODIPY® FL C5-ceramide, BODIPY® TR ceramide, BODIPY® FL C5-ganglioside GM1 and BODIPY® FL C5-lactosylceramide, each complexed with defatted BSA (B22650, B34400, B34401, B34402, respectively). BODIPY® FL C5-ceramide has been used to investigate the linkage of sphingolipid metabolism to protein secretory pathways53–56 and neuronal growth.47,57 Internalization of BODIPY® FL C5sphingomyelin from the plasma membrane of human skin fibroblasts results in a mixed population of labeled endosomes that can be distinguished based on the concentration-dependent green Figure 13.3.7 Cells in the notochord rudiment of a zebrafish embryo undergoing mediolateral intercalation to lengthen the forming notochord. BODIPY® FL C5-ceramide (D3521) localizes in the interstitial fluid of the zebrafish embryo and freely diffuses between cells, illuminating cell boundaries. This confocal image was obtained using a Bio-Rad® MRC-600 microscope. Image contributed by Mark Cooper, University of Washington. Figure 13.3.8 Nucleus and Golgi apparatus of a bovine pulmonary artery endothelial cell (BPAEC) labeled with Hoechst 33342 (H1399, H3569, H21492) and the BSA complex of BODIPY® FL C5-ceramide (D3521, B22650), respectively. Figure 13.3.5 N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-s-indacene-3-pentanoyl)sphingosyl phosphocholine (BODIPY® FL C5-sphingomyelin, D3522). Figure 13.3.6 Selective staining of the Golgi apparatus using the green-fluorescent BODIPY® FL C5-ceramide (D3521) (top). At high concentrations, the BODIPY® FL fluorophore forms excimers that can be visualized using a red longpass optical filter (bottom). The BODIPY® FL C5-ceramide accumulation in the trans-Golgi is sufficient for excimer formation (J Cell Biol (1991) 113:1267). Images contributed by Richard Pagano, Mayo Foundation. Figure 13.3.9 BODIPY® TR ceramide (N-((4-(4,4-difluoro5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy) acetyl)sphingosine; D7540). ™ The Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies TheMolecular Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPORTANT NOTICE:described The products described this covered manual are by oneLimited or moreUse Limited Use Label License(s). to the Appendix IMPORTANT NOTICE : The products in this manualinare by covered one or more Label License(s). PleasePlease referrefer to the Appendix on on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes 567 Chapter 13 — Probes for Lipids and Membranes Section 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes (~515 nm) or red (~620 nm) emission of the probe31 (Figure 13.3.6). BODIPY® C5-sphingomyelin has also been used to assess sphingomyelinase gene transfer and expression in hematopoietic stem and progenitor cells.4 Studies by Martin and Pagano have shown that the internalization routes for BODIPY® FL C5-glucocerebroside follow both endocytic and nonendocytic pathways and are quite different from those for BODIPY® FL C5-sphingomyelin.58 BODIPY® FL C5-lactosylceramide, BODIPY® FL C5-ganglioside GM1 and BODIPY® FL cerebrosides are useful tools for the study of glycosphingolipid transport and signaling pathways in cells59,60 and for diagnosis of lipid-storage disorders such as Niemann–Pick disease,61 Gaucher disease, GM1 gangliosidosis, Morquio syndrome and type IV mucolipidosis 6,49,62–67 (MLIV). Addition of BODIPY® FL C5-lactosylceramide to the culture medium of cells from patients with sphingolipid-storage diseases (sphingolipidosis) results in fluorescent product accumulation in lysosomes, whereas this probe accumulates in the Golgi apparatus of normal cells and cells from patients with other storage diseases.46,48 BODIPY® FL C5-ganglioside GM1 has been shown to form cholesterol-enhanced clusters in membrane complexes with amyloid β-protein in a model of Alzheimer disease amyolid fibrils.68 As observed by fluorescence microscopy, the colocalization of BODIPY® FL C5-ganglioside GM1 and fluorescent cholera toxin B conjugates (Section 7.7) provides a direct indication of the association of these molecules in lipid rafts50 (Figure 13.3.10). NBD Sphingolipids Figure 13.3.10 A J774 mouse macrophage cell sequentially stained with BODIPY® FL ganglioside GM1 (B13950) and then with Alexa Fluor® 555 dye–labeled cholera toxin subunit B (C22843, C34776; also available as a component of V34404). The cell was then treated with an anti–CT-B antibody (a component of V34404) to induce crosslinking. Alexa Fluor® 555 dye fluorescence (top panel, red) and BODIPY® FL dye fluorescence (middle panel, green) were imaged separately and overlaid to emphasize the coincident staining (bottom panel, yellow). Nuclei were stained with blue-fluorescent Hoechst 33342 (H1399, H3570, H21492). Figure 13.3.11 NBD C6-ceramide (6-((N-(7-nitrobenz-2-oxa1,3-diazol-4-yl)amino)hexanoyl)sphingosine, N1154). NBD C6 -ceramide (N1154, Figure 13.3.11) and NBD C6 -sphingomyelin (N3524) analogs predate their BODIPY® counterparts and have been extensively used for following sphingolipid metabolism in cells9,59,69,70 and in multicellular organisms.71 As with BODIPY® FL C5-ceramide, we also offer NBD C6 -ceramide complexed with defatted BSA (N22651) to facilitate cell loading without the use of organic solvents to dissolve the probe. Koval and Pagano have prepared NBD analogs of both the naturally occurring D-erythro and the nonnatural L-threo stereoisomers of sphingomyelin and have compared their intracellular transport behavior in Chinese hamster ovary (CHO) fibroblasts.72 NBD C6 -ceramide lacks the useful concentration-dependent optical properties of the BODIPY® FL analog and is less photostable; however, the fluorescence of NBD C 6 -ceramide is apparently sensitive to the cholesterol content of the Golgi apparatus, a phenomenon that is not observed with BODIPY® FL C5-ceramide. If NBD C6 -ceramide–containing cells are starved for cholesterol, the NBD C6 -ceramide that accumulates within the Golgi apparatus appears to be severely photolabile but this NBD photobleaching can be reduced by stimulation of cholesterol synthesis. Thus, NBD C6 -ceramide may be useful in monitoring the cholesterol content of the Golgi apparatus in live cells.73 Vybrant® Lipid Raft Labeling Kits The Vybrant® Lipid Raft Labeling Kits (V34403, V34404, V34405) are designed to provide convenient, reliable and extremely bright fluorescent labeling of lipid rafts in live cells. Lipid rafts are detergent-insoluble, sphingolipid- and cholesterol-rich membrane microdomains that form lateral assemblies in the plasma membrane.19–25 Lipid rafts also sequester glycophosphatidylinositol (GPI)-linked proteins and other signaling proteins and receptors, which may be regulated by their selective interactions with these membrane microdomains.50,74–78 Lipid rafts play a role in a variety of cellular processes—including the compartmentalization of cell-signaling events,79–86 the regulation of apoptosis87–89 and the intracellular trafficking of certain membrane proteins and lipids90–92—as well as in the infectious cycles of several viruses and bacterial pathogens.93–98 Examining the formation and regulation of lipid rafts is a critical step in understanding these aspects of eukaryotic cell function. The Vybrant® Lipid Raft Labeling Kits provide the key reagents for fluorescently labeling lipid rafts in vivo with our bright and extremely photostable Alexa Fluor® dyes (Figure 13.3.10). Live cells are first labeled with the green-fluorescent Alexa Fluor® 488, orange-fluorescent Alexa Fluor® 555 or red-fluorescent Alexa Fluor® 594 conjugate of cholera toxin subunit B (CT-B). This CT-B conjugate binds to the pentasaccharide chain of plasma membrane ganglioside GM1, which selectively partitions into lipid rafts.50,99,100 All Molecular Probes® CT-B conjugates are prepared from recombinant CT-B and are completely free of the toxic subunit A, thus eliminating any concern for toxicity or ADP-ribosylating activity. An antibody that specifically recognizes CT-B is then TheMolecular MolecularProbes® Probes Handbook: Handbook: AAGuide and Labeling Labeling Technologies Technologies The GuidetotoFluorescent Fluorescent Probes Probes and ™ 568 IMPORTANT NOTICE: The products described in this manual covered one or more Limited Use Label License(s). Please refer to thePlease Appendix IMPORTANT NOTICE : The products described in thisare manual arebycovered by one or more Limited Use Label License(s). referonto page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes Chapter 13 — Probes for Lipids and Membranes Section 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes used to crosslink the CT-B–labeled lipid rafts into distinct patches on the plasma membrane, which are easily visualized by fluorescence microscopy.101,102 Each Vybrant® Lipid Raft Labeling Kit contains sufficient reagents to label 50 live-cell samples in a 2 mL assay, including: • Recombinant cholera toxin subunit B (CT-B) labeled with the Alexa Fluor® 488 (in Kit V34403), Alexa Fluor® 555 (in Kit V34404) or Alexa Fluor® 594 (in Kit V34405) dye • Anti–cholera toxin subunit B antibody (anti–CT-B) • Concentrated phosphate-buffered saline (PBS) • Detailed labeling protocols Because they are compatible with various multilabeling schemes, the Vybrant® Lipid Raft Labeling Kits can also serve as important tools for identifying physiologically significant membrane proteins that associate with lipid rafts. Cells can be labeled with other live-cell probes during the lipid raft labeling protocol or immediately following the antibody crosslinking step, depending on the specific labeling requirements of the other probes. Alternatively, once the lipid rafts have been labeled and crosslinked, the cells can be fixed for long-term storage or fixed and permeabilized for subsequent labeling with antibodies or other probes that are impermeant to live cells. Amplex® Red Sphingomyelinase Assay Kit The Amplex® Red Sphingomyelinase Assay Kit (A12220) is designed for measuring sphingomyelinase activity in solution using a fluorescence microplate reader or fluorometer103 (Figure 13.3.12). This assay should be useful for screening sphingomyelinase activators or inhibitors or for detecting sphingomyelinase activity in cell and tissue extracts. The assay, which uses natural sphingomyelin as the principal substrate, employs an enzyme-coupled detection scheme in which phosphocholine liberated by the action of sphingomyelinase is cleaved by alkaline phosphatase to generate choline. Choline is, in turn, oxidized by choline oxidase, generating H2O2, which drives the conversion of the Amplex® Red reagent (A12222, A22177; Section 10.5) to red-fluorescent resorufin. This sensitive assay technique has been employed to detect activation of acid sphingomyelinase associated with ultraviolet • • • • • • • • • • • Amplex® Red reagent Dimethylsulfoxide (DMSO) Horseradish peroxidase (HRP) H2O2 for use as a positive control Concentrated reaction buffer Choline oxidase from Alcaligenes sp. Alkaline phosphatase from calf intestine Sphingomyelin Triton X-100 Sphingomyelinase from Bacillus sp. Detailed protocols Each kit provides sufficient reagents for approximately 500 assays using a fluorescence microplate reader and a reaction volume of 200 µL per assay. Steroids Most steroids are neutral lipids and, as such, localize primarily within the cell’s membranes, in lipid vacuoles and bound to certain lipoproteins. Fluorescent analogs of these biomolecules, most of which are derived from BODIPY® and NBD dyes, are highly lipophilic probes. One application of these probes is to detect enzymatic activity—either in vitro or in vivo—through hydrolysis of the fatty acid esters to fluorescent fatty acids.106 Although the substrates and products in these enzyme assays typically have similar fluorescence properties, they are readily extracted by an organic solvent and separated by chromatography. We have also developed sensitive fluorometric assays for cholesterol, cholesteryl esters and enzymes that metabolize natural cholesterol derivatives; the assay reagents and protocols are available in our Amplex® Red Cholesterol Assay Kit (A12216) described below. A review of the cellular organization, functions and transport of cholesterol has recently been published.107 BODIPY® Cholesteryl Esters 6000 Cholesteryl esters consist of a fatty acid esterified to the 3β-hydroxyl group of cholesterol (Figure 13.3.13). These very nonpolar species are the predominant lipid components of atherosclerotic plaque and lowand high-density lipoprotein (LDL and HDL) cores. We offer cholesteryl esters of three of our BODIPY® fatty acids—BODIPY® FL C12 (C3927MP), BODIPY® 542/563 C11 (C12680) and BODIPY® 576/589 C11 (C12681)—all of which have long-wavelength visible emission. BODIPY® 5000 Fluorescence radiation–induced apoptosis104 and to characterize an insecticidal sphingomyelinase C produced by Bacillus cereus.105 The Amplex® Red Sphingomyelinase Assay Kit contains: 4000 1000 3000 800 600 2000 400 1000 200 0 0 0 10 20 0 0.2 30 0.4 40 0.6 50 Sphingomyelinase (mU/mL) Figure 13.3.12 Measurement of sphingomyelinase activity using the Amplex® Red Sphingomyelinase Assay Kit (A12220). Each reaction contained 50 µM Amplex® Red reagent, 1 U/mL horseradish peroxidase (HRP), 0.1 U/mL choline oxidase, 4 U/mL of alkaline phosphatase, 0.25 mM sphingomyelin and the indicated amount of Staphylococcus aureus sphingomyelinase in 1X reaction buffer. Reactions were incubated at 37°C for one hour. Fluorescence was measured with a fluorescence microplate reader using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm. Figure 13.3.13 Cholesteryl BODIPY® FL C12 (cholesteryl 4,4-difluoro-5,7-dimethyl-4-bora3a,4a-diaza-s-indacene-3-dodecanoate; C3927MP). ™ The Handbook: A Guide to Fluorescent Probes and Labeling Technologies TheMolecular MolecularProbes Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPORTANT NOTICE:described The products described this covered manual are by oneLimited or moreUse Limited UseLicense(s). Label License(s). Please to the Appendix IMPORTANT NOTICE : The products in this manualin are bycovered one or more Label Please referrefer to the Appendix on on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes 569 Chapter 13 — Probes for Lipids and Membranes Section 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes FL cholesteryl ester can be used as a tracer of cholesterol transport and receptor-mediated endocytosis of lipoproteins by fluorescence microscopy108–110 (Figure 13.3.14) and as a general nonexchangeable membrane marker. Addition of methyl β-cyclodextrin to BODIPY® FL cholesteryl ester is reported to facilitate its uptake by cells and tissues.111 Researchers have extensively used BODIPY® FL cholesteryl ester to measure cholesteryl ester–transfer protein (CETP) activity using fluorescence microplate readers.112–115 The longer-wavelength BODIPY® 542/563 and BODIPY® 576/589 cholesteryl esters likely have similar applications. Side Chain–Modified Cholesterol Analog We offer an NBD-labeled cholesterol analog in which the fluorophore replaces the terminal segment of cholesterol’s flexible alkyl tail. The environment-sensitive NBD fluorophore of the NBD cholesterol analog (N1148) localizes in the membrane’s interior, unlike the anomalous positioning of NBD-labeled phospholipid acyl chains.116 As with other NBD lipid analogs, this probe is useful for investigating lipid transport processes117,118 and lipid–protein interactions.119,120 NBD cholesterol is selectively taken up by high-density lipoproteins via the scavenger receptor B1.117 A lipid droplet–specific protein binds unesterified NBD cholesterol with extremely high affinity117 (Kd = 2 nM). Figure 13.3.14 Selective uptake of cholesteryl esters (CE) in rat ovarian granulosa cells as monitored with cholesteryl BODIPY® FL C12 (C3927MP). The hormone-stimulated cells internalized and stored CEs derived from reconstituted high-density lipoprotein (HDL)–BODIPY® CE complexes (J Biol Chem (1996) 271:16208). A low-light (<100 µW beam power) computerized imaging system minimized any photobleaching of the fluorophore. This pseudocolored image uses yellow-green to illustrate the low-level fluorescence of the cytoplasmic membranes, yellow to illustrate the medium-level fluorescence of the Golgi, and red to illustrate the high-level fluorescence of the lipid droplets. Image contributed by Eve Reaven, VA Medical Center, Palo Alto, California. 6000 Fluorescence 5000 Amplex® Red Cholesterol Assay Kit 4000 3000 The Amplex® Red Cholesterol Assay Kit (A12216) provides an exceptionally sensitive assay for both cholesterol and cholesteryl esters in complex mixtures and is suitable for use with either fluorescence microplate readers or fluorometers. The assay provided in this kit is designed to detect as little as 5 ng/mL (5 × 10 –4 mg/dL) cholesterol (Figure 13.3.15) and to accurately measure the cholesterol or cholesteryl ester content in the equivalent of 0.01 µL of human serum.121 The assay uses an enzyme-coupled reaction scheme in which cholesteryl esters are hydrolyzed by cholesterol esterase into cholesterol, which is then oxidized by cholesterol oxidase to yield H2O2 and the corresponding ketone steroidal product (Figure 13.3.16). The H2O2 is then detected using the Amplex® Red reagent in combination with horseradish peroxidase (HRP). 30 20 2000 10 1000 0 0 0 1 2 0 0.005 0.010 0.015 3 4 Cholesterol (µg/mL) Figure 13.3.15 Detection of cholesterol using the Amplex® Red Cholesterol Assay Kit (A12220). Each reaction contained 150 µM Amplex® Red reagent, 1 U/mL horseradish peroxidase (HRP), 1 U/mL cholesterol oxidase, 1 U/mL cholesterol esterase and the indicated amount of cholesterol in 1X reaction buffer. Reactions were incubated at 37°C for 30 minutes. Fluorescence was measured with a fluorescence microplate reader using excitation at 560 ± 10 nm and fluorescence detection at 590 ± 10 nm. The insert above shows the high sensitivity and excellent linearity of the assay at low cholesterol levels (0–10 ng/mL). Figure 13.3.16 Enzyme-coupled Amplex® Red assays. Enzyme reactions that produce H2O2 can be made into Amplex® Red assays. The Amplex® Red Cholesterol Assay Kit (A12216) uses cholesterol oxidase to produce H2O2, which is then detected by the Amplex® Red reagent in the presence of horseradish peroxidase (HRP). Similarly, the Amplex® Red Acetylcholine/Acetylcholinesterase Assay Kit (A12217) uses choline oxidase to produce H2O2. The MolecularProbes® Probes Handbook: Handbook: A Probesand andLabeling LabelingTechnologies Technologies The Molecular A Guide Guide to to Fluorescent Fluorescent Probes ™ 570 IMPORTANT NOTICE: The products described in this manual aremanual coveredare by one or more Use Label License(s). Please refer to thePlease Appendix on to IMPORTANT NOTICE : The products described in this covered by Limited one or more Limited Use Label License(s). refer page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes Chapter 13 — Probes for Lipids and Membranes Section 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes The Amplex® Red cholesterol assay is continuous and requires no separation or wash steps. These characteristics make the assay particularly well suited for the rapid and direct analysis of cholesterol in blood and food samples using automated instruments. By performing two separate measurements in the presence and absence of cholesterol esterase, this assay is also potentially useful for determining the fraction of cholesterol that is in the form of cholesteryl esters within a sample. In addition, by adding an excess of cholesterol to the reaction, this assay can be used to sensitively detect the activity of cholesterol oxidase. The Amplex® Red Cholesterol Assay Kit contains: • • • • • • • • • Amplex® Red reagent Dimethylsulfoxide (DMSO) Horseradish peroxidase (HRP) H2O2 for use as a positive control Concentrated reaction buffer Cholesterol oxidase from Streptomyces Cholesterol esterase from Pseudomonas Cholesterol for preparation of a standard curve Detailed protocols Figure 13.3.17 1,2-Dioleoyl-3-(1-pyrenedodecanoyl)-rac-glycerol (D6562). Fluorescence emission Each kit provides sufficient reagents for approximately 500 assays using a fluorescence microplate reader and a reaction volume of 100 µL per assay. Fluorescent Triacylglycerol The fluorescent triacylglycerol 1,2-dioleoyl-3-(1-pyrenedodecanoyl)rac-glycerol (D6562) has a pyrene fatty acid ester replacing one of the three fatty acyl residues of a natural triacylglycerol (Figure 13.3.17). Pyrene has the important spectral property of forming excimers (Figure 13.3.18) when two fluorophores are in close proximity during the excited state. Pyrene triacylglycerols are useful for measuring cholesteryl ester transfer protein–mediated triacylglycerol transport between plasma lipoproteins.122 They are also excellent substrates for lipoprotein lipase and hepatic triacylglycerol lipase.123 1 2 3 4 350 400 450 500 550 600 Wavelength (nm) Figure 13.3.18 Excimer formation by pyrene in ethanol. Spectra are normalized to the 371.5 nm peak of the monomer. All spectra are essentially identical below 400 nm after normalization. Spectra are as follows: 1) 2 mM pyrene, purged with argon to remove oxygen; 2) 2 mM pyrene, air-equilibrated; 3) 0.5 mM pyrene (argon-purged); and 4) 2 µM pyrene (argonpurged). The monomer-to-excimer ratio (371.5 nm/470 nm) is dependent on both pyrene concentration and the excited-state lifetime, which is variable because of quenching by oxygen. Lipopolysaccharides Fluorescent Lipopolysaccharides 64 • • • • • Alexa Fluor® 488 LPS from E. coli serotype 055:B5 (L23351) Alexa Fluor® 488 LPS from S. minnesota (L23356) Alexa Fluor® 568 LPS from E. coli serotype 055:B5 (L23352) Alexa Fluor® 594 LPS from E. coli serotype 055:B5 (L23353) BODIPY® FL LPS from E. coli serotype 055:B5 (L23350) LPS, also known as endotoxins, are a family of complex glycolipid molecules located on the surface of gram-negative bacteria. LPS play a large role in protecting the bacterium from host defense mechanisms and antibiotics. Binding of LPS to the CD14 cell-surface receptor of phagocytes (Figure 13.3.19) is the key initiation step in the mammalian immune response to infection by gram-negative bacteria. The structural Unlabeled cells Events We offer fluorescent conjugates of lipopolysaccharides (LPS) from Escherichia coli and Salmonella minnesota (Section 16.1, Table 16.1), including: Labeled cells 0 100 101 102 103 104 Alexa Fluor® 488 LPS fluorescence Figure 13.3.19 Flow cytometry analysis of blood using an Alexa Fluor® 488 lipopolysaccharide (LPS). Human blood was incubated with Alexa Fluor® 488 LPS from Escherichia coli (L23351) and anti-CD14 antibody on ice for 20 minutes. The red blood cells were lysed and the sample was analyzed on a flow cytometer equipped with a 488 nm Ar-Kr excitation source and a 525 ± 12 nm bandpass emission filter. Monocytes were identified based on their light scatter and CD14 expression. ™ The Handbook: A Guide to Fluorescent Probes and Labeling Technologies TheMolecular MolecularProbes Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPORTANT NOTICE:described The products described this covered manual are by oneLimited or moreUse Limited UseLicense(s). Label License(s). to the Appendix IMPORTANT NOTICE : The products in this manualin are bycovered one or more Label PleasePlease referrefer to the Appendix on on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes 571 Chapter 13 — Probes for Lipids and Membranes OH O O HO P OH O O O O O O NH HO NH O O P OH OH O O O O O O O HO O O Figure 13.3.20 Structure of the lipid A component of Salmonella minnesota lipopolysaccharide. Section 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes core of LPS, and the primary determinant of its biological activity, is an N-acetylglucosamine derivative, lipid A (Figure 13.3.20). Two plasma proteins, LPS-binding protein (LBP) and soluble CD14 (sCD14), play primary roles in transporting LPS and mediating cellular responses.124–129 If the fatty acid residues are removed from the lipid A component, the toxicity of the LPS can be reduced significantly; however, the mono- or diphosphoryl forms of lipid A are inherently toxic. In many gram-negative bacterial infections, LPS are responsible for clinically significant symptoms like fever, low blood pressure and tissue edema, which can lead to disseminated intravascular coagulation, organ failure and death. Studies also clearly indicate that LPS induce various signal transduction pathways, including those involving protein kinase C130,131 and protein myristylation,132 and stimulate a variety of immunochemical responses, including B lymphocyte133 and G-protein activation.134 The fluorescent BODIPY® FL and Alexa Fluor® LPS conjugates, which are labeled with succinimidyl esters of these dyes, allow researchers to follow LPS binding, transport and cell internalization processes. Lipopolysaccharide internalization activates endotoxin-dependent signal transduction in cardiomyocytes.135 The Alexa Fluor® 488 LPS conjugates (L23351, L23356) selectively label microglia in a mixed culture containing oligodendrocyte precursors, astrocytes and microglia.136 A biologically active conjugate of galactose oxidase–oxidized S. minnesota LPS and our Alexa Fluor® 488 hydrazide (A10436, Section 3.3; A10440) has been used to elucidate molecular mechanisms of septic shock.137 The BODIPY® FL derivative of LPS from E. coli strain LCD25 (L23350) was used to measure the transfer rate of LPS from monocytes to high-density lipoprotein138 (HDL). Another study utilized a BODIPY® FL derivative of LPS from S. minnesota to demonstrate transport to the Golgi apparatus in neutrophils,124,125 although this could have been due to probe metabolism. It has been reported that organelles other than the Golgi are labeled by some fluorescent or nonfluorescent LPS.139,140 Cationic lipids are reported to assist the translocation of fluorescent lipopolysaccharides into live cells;141 cell surface–bound LPS can be quenched by trypan blue.138 Molecular Probes® fluorescent LPS can potentially be combined with other fluorescent indicators, such as Ca 2+-, pH- or organelle-specific stains, for monitoring intracellular localization and real-time changes in cellular response to LPS. Pro-Q® Emerald 300 Lipopolysaccharide Gel Stain Kit Fluorescence Fluorescence Figure 13.3.21 Lipopolysaccharide staining with the Pro-Q® Emerald 300 Lipopolysaccharide Gel Stain Kit. Lipopolysaccharides (LPS) were electrophoresed through a 13% acrylamide gel and stained using the Pro-Q® Emerald 300 Lipopolysaccharide Gel Stain Kit (P20495). From left to right, the lanes contain: CandyCane™ glycoprotein molecular weight standards (~250 ng/band), blank, 4, 1 and 0.25 µg of LPS from Escherichia coli smooth serotype 055:B5 and 4, 1 and 0.25 µg of LPS from E. coli rough mutant EH100 (Ra mutant). The Pro-Q® Emerald 300 Lipopolysaccharide Gel Stain Kit (P20495) provides a simple, rapid and highly sensitive method for staining lipopolysaccharides (LPS) in gels (Figure 13.3.21, Figure 13.3.22, Figure 13.3.23). The structure of this important class of molecules can be analyzed by SDS-polyacrylamide gel electrophoresis, during which the heterogeneous mixture of polymers separates into a characteristic ladder pattern. This ladder has conventionally been detected using silver staining.142–144 However, despite the long and complex procedures required, silver staining provides poor sensitivity and cannot differentiate LPS from proteins in the sample. An alternative 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Figure 13.3.22 Characterization of lipopolysaccharides. Lipopolysaccharides (LPS) from Escherichia coli smooth serotype 055:B5 were loaded onto a 13% polyacrylamide gel. Following electrophoresis, the gel was stained using the Pro-Q® Emerald 300 Lipopolysaccharide Gel Stain Kit (P20495), and the fluorescence was measured for the lane. A plot of fluorescence signal versus the relative distance from the dye front shows a characteristic laddering profile for smooth-type LPS. ™ IMPORTANT NOTICE: The products described in this manual covered bycovered one or more Limited Use Label License(s). Please refer to thePlease Appendix IMPORTANT NOTICE : The products described in thisaremanual are by one or more Limited Use Label License(s). referonto page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. 2 3 4 Figure 13.3.23 Linearity of the Pro-Q® Emerald 300 stain for lipopolysaccharide (LPS) detection. A dilution series of lipopolysaccharides from Escherichia coli smooth serotype 055:B5 was loaded onto a 13% polyacrylamide gel. Following electrophoresis, the gel was stained using the Pro-Q® Emerald 300 Lipopolysaccharide Gel Stain Kit (P20495) and the same band from each lane was quantitated using a CCD camera. A plot of the fluorescence intensity versus the mass of LPS loaded shows a linear range over two orders of magnitude. The MolecularProbes® Probes Handbook: Handbook: AAGuide and Labeling LabelingTechnologies Technologies The Molecular Guideto toFluorescent Fluorescent Probes Probes and 572 1 Mass of LPS (µg) Migration (Rf) the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes Chapter 13 — Probes for Lipids and Membranes Section 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes staining method that makes use of the reaction of the carbohydrates with detectable hydrazides obtains higher sensitivity, but requires blotting to a membrane and time- and labor-intensive procedures.145–149 By comparison, the staining technology used in the Pro-Q® Emerald 300 Lipopolysaccharide Gel Stain Kit vastly simplifies detection of LPS in SDS-polyacrylamide gels. The key to this novel methodology is our bright green-fluorescent Pro-Q® Emerald 300 dye, which covalently binds to periodate-oxidized carbohydrates of LPS. This dye allows the detection of as little as 200 pg of LPS in just a few hours using a simple UV transilluminator. The sensitivity is at least 50–100 times that of silver staining and requires much less hands-on time. This dye is also used in our Pro-Q® Emerald 300 and Multiplexed Proteomics® Glycoprotein Stain Kits (P21855, P21857, M33307; Section 9.4) and may be useful for detection of other molecules containing carbohydrates or aldehydes. The Pro-Q® Emerald 300 Lipopolysaccharide Gel Stain Kit contains: • • • • • Pro-Q® Emerald 300 reagent Pro-Q® Emerald 300 staining buffer Oxidizing reagent (periodic acid) Smooth LPS standard from Escherichia coli serotype 055-B5 Detailed protocols Sufficient materials are supplied to stain ten 8 cm × 10 cm gels, 0.5–0.75 mm thick. REFERENCES 1. Annu Rev Biochem (1998) 67:27; 2. Prog Lipid Res (1997) 36:153; 3. Biochim Biophys Acta (1998) 1436:233; 4. Blood (1999) 93:80; 5. Biochim Biophys Acta (1984) 793:169; 6. Clin Chim Acta (1982) 124:123; 7. Clin Chim Acta (1984) 142:313; 8. J Cell Biol (1990) 111:429; 9. Methods Cell Biol (1993) 38:221; 10. J Biol Chem (1993) 268:17762; 11. Cell Signal (1998) 10:685; 12. Trends Biochem Sci (1995) 20:73; 13. Curr Opin Oncol (1998) 10:552; 14. J Inherit Metab Dis (1998) 21:472; 15. Science (1993) 259:1769; 16. Ann N Y Acad Sci (1998) 845:139; 17. Ann N Y Acad Sci (1998) 845:57; 18. J Neurochem (1998) 70:1335; 19. J Cell Biol (2003) 162:365; 20. J Lipid Res (2003) 44:655; 21. Eur J Biochem (2002) 269:737; 22. Science (2000) 290:1721; 23. Mol Membr Biol (1999) 16:145; 24. Trends Cell Biol (1999) 9:87; 25. Annu Rev Cell Dev Biol (1998) 14:111; 26. Biochemistry (2001) 40:4893; 27. Trends Cell Biol (2000) 10:408; 28. J Biol Chem (1994) 269:3125; 29. Chem Phys Lipids (1999) 102:55; 30. Ann N Y Acad Sci (1998) 845:152; 31. Biophys J (1997) 72:37; 32. J Cell Biol (1998) 140:39; 33. Histochem Cell Biol (2008) 130:819; 34. Methods Enzymol (2000) 312:293; 35. Methods Enzymol (2000) 312:523; 36. Methods (2005) 36:186; 37. J Cell Biol (1996) 134:1031; 38. J Cell Biol (1991) 113:1267; 39. Cytometry (1993) 14:251; 40. J Cell Biol (1993) 120:399; 41. Eur J Cell Biol (1992) 58:214; 42. Mol Biochem Parasitol (2000) 106:21; 43. Infect Immun (2000) 68:5960; 44. J Cell Biol (2001) 154:535; 45. Am J Physiol Lung Cell Mol Physiol (2001) 280:L938; 46. Nat Cell Biol (1999) 1:386; 47. J Neurochem (1999) 73:1375; 48. Lancet (1999) 354:901; 49. Proc Natl Acad Sci U S A (1998) 95:6373; 50. J Cell Biol (1999) 147:447; 51. J Cell Biol (2002) 157:327; 52. Biophys J (1999) 77:1498; 53. J Cell Sci (2006)119:2084; 54. Mol Biol Cell (1995) 6:135; 55. J Biol Chem (1993) 268:4577; 56. Biochemistry (1992) 31:3581; 57. J Biol Chem (1993) 268:14476; 58. J Cell Biol (1994) 125:769; 59. Biochim Biophys Acta (1992) 1113:277; 60. Brain Res (1992) 597:108; 61. Anal Biochem (2001) 293:204; 62. Biochim Biophys Acta (1999) 1455:85; 63. Traffic (2000) 1:807; 64. J Biol Chem (1993) 268:14861; 65. Biochim Biophys Acta (1987) 915:87; 66. Biochem Biophys Res Comm (1965) 18:221; 67. Anal Biochem (1984) 136:223; 68. J Biol Chem (2001) 276:24985; 69. Adv Cell Mol Biol Membranes (1993) 1:199; 70. Biochim Biophys Acta (1991) 1082:113; 71. Parasitology (1992) 105:81; 72. J Cell Biol (1989) 108:2169; 73. Proc Natl Acad Sci U S A (1993) 90:2661; 74. Proc Natl Acad Sci U S A (2003) 100:5813; 75. J Immunol (2003) 170:1329; 76. J Membr Biol (2002) 189:35; 77. Proc Natl Acad Sci U S A (2001) 98:9098; 78. Mol Biol Cell (1999) 10:3187; 79. Biochim Biophys Acta (2003) 1610:247; 80. Annu Rev Immunol (2003) 21:457; 81. Mol Immunol (2002) 38:1247; 82. Nat Rev Immunol (2002) 2:96; 83. Biol Res (2002) 35:127; 84. Nat Rev Mol Cell Biol (2000) 1:31; 85. J Exp Med (1999) 190:1549; 86. J Cell Biol (1998) 143:637; 87. Immunity (2003) 18:655; 88. J Biol Chem (2002) 277:39541; 89. Biochem Biophys Res Commun (2002) 297:876; 90. Biol Chem (2002) 383:1475; 91. J Cell Biol (2001) 153:529; 92. J Cell Sci (2001) 114:3957; 93. J Virol (2003) 77:9542; 94. Exp Cell Res (2003) 287:67; 95. Traffic (2002) 3:705; 96. J Clin Virol (2001) 22:217; 97. Curr Biol (2000) 10:R823; 98. J Virol (2000) 74:3264; 99. Biochemistry (1996) 35:16069; 100. Mol Microbiol (1994) 13:745; 101. J Cell Biol (1998) 141:929; 102. J Biol Chem (1994) 269:30745; 103. Am J Pathol (2002) 161:1061; 104. J Biol Chem (2001) 276:11775; 105. Eur J Biochem (2004) 271:601; 106. J Lipid Res (1995) 36:1602; 107. Nat Rev Mol Cell Biol (2008) 9:125; 108. Proc Natl Acad Sci U S A (2001) 98:1613; 109. J Biol Chem (1997) 272:25283; 110. PLoS ONE (2007) 2:e511; 111. Am J Physiol (1999) 277:G1017; 112. Biochemistry (1995) 34:12560; 113. Chem Phys Lipids (1995) 77:51; 114. Lipids (1994) 29:811; 115. J Lipid Res (1993) 34:1625; 116. J Phys Chem (1999) 103:8180; 117. J Biol Chem (2000) 275:12769; 118. J Lipid Res (1999) 40:1747; 119. Biochim Biophys Acta (1999) 1437:37; 120. J Biol Chem (1999) 274:35425; 121. J Biochem Biophys Methods (1999) 38:43; 122. J Biochem (Tokyo) (1998) 124:237; 123. Lipids (1988) 23:605; 124. J Exp Med (1999) 190:523; 125. J Exp Med (1999) 190:509; 126. J Biol Chem (1996) 271:4100; 127. J Exp Med (1995) 181:1743; 128. J Exp Med (1994) 180:1025; 129. J Exp Med (1994) 179:269; 130. J Biol Chem (1984) 259:10048; 131. J Exp Med (1996) 183:1899; 132. Proc Natl Acad Sci U S A (1986) 83:5817; 133. Adv Immunol (1979) 28:293; 134. Eur J Immunol (1989) 19:125; 135. Circ Res (2001) 88:491; 136. J Neurosci (2002) 22:2478; 137. Cytometry (2000) 41:316; 138. J Biol Chem (1999) 274:34116; 139. Electron Microsc Rev (1992) 5:381; 140. J Periodontol (1985) 56:553; 141. Biotechniques (2000) 28:510; 142. J Clin Microbiol (1990) 28:2627; 143. Microbiol Immunol (1991) 35:331; 144. J Biochem Biophys Methods (1993) 26:81; 145. Electrophoresis (1998) 19:2398; 146. Appl Environ Microbiol (1995) 61:2845; 147. Electrophoresis (1999) 20:462; 148. Electrophoresis (2000) 21:526; 149. Anal Biochem (1990) 188:285. DATA TABLE 13.3 SPHINGOLIPIDS, STEROIDS, LIPOPOLYSACCHARIDES AND RELATED PROBES Cat. No. B13950 B22650 B34400 B34401 B34402 C3927MP C12680 C12681 D3521 D3522 D6562 D7519 D7540 MW 1582.50 ~66,000 ~66,000 ~66,000 ~66,000 786.98 851.02 809.97 601.63 766.75 1003.54 861.96 705.71 Storage F,D,L F,D,L F,D,L F,D,L F,D,L F,D,L F,D,L F,D,L FF,D,L FF,D,L FF,D,L,A FF,D,L FF,D,L Soluble DMSO, EtOH H2O H2O H2O H2O CHCl3 CHCl3 CHCl3 CHCl3, DMSO see Notes CHCl3 DMSO, EtOH CHCl3, DMSO Abs 505 505 589 505 505 505 543 579 505 505 341 505 589 EC 80,000 91,000 65,000 80,000 80,000 86,000 57,000 98,000 91,000 77,000 40,000 85,000 65,000 Em 512 511 616 512 511 511 563 590 511 512 376 511 616 Solvent MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH Notes 1 1, 2 2 1, 2 1, 2 3 3 3 1 1, 4 5, 6 1 continued on next page ™ The Handbook: A Guide to Fluorescent Probes and Labeling Technologies TheMolecular MolecularProbes Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes 573 Chapter 13 — Probes for Lipids and Membranes Section 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes DATA TABLE 13.3 SPHINGOLIPIDS, STEROIDS, LIPOPOLYSACCHARIDES AND RELATED PROBES—continued Cat. No. MW Storage Soluble Abs EC Em Solvent Notes D7711 864.94 FF,D,L DMSO 505 75,000 513 MeOH 1, 7 D13951 925.91 FF,D,L DMSO, EtOH 505 80,000 511 MeOH 1 469 21,000 537 MeOH 8 N1148 494.63 L CHCl3, MeCN 466 22,000 536 MeOH 8 N1154 575.75 FF,D,L CHCl3, DMSO N3524 740.88 FF,D,L see Notes 466 22,000 536 MeOH 4, 8 466 22,000 536 MeOH 2, 8 N22651 ~66,000 F,D,L H2O For definitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages. Notes 1. Em for BODIPY® FL sphingolipids shifts to ~620 nm when high concentrations of the probe (>5 mol %) are incorporated in lipid mixtures. (J Cell Biol (1991) 113:1267) 2. This product is a lipid complexed with bovine serum albumin (BSA). Spectroscopic data are for the free lipid in MeOH. 3. The absorption and fluorescence spectra of BODIPY® derivatives are relatively insensitive to the solvent. 4. Chloroform is the most generally useful solvent for preparing stock solutions of phospholipids (including sphingomyelins). Glycerophosphocholines are usually freely soluble in ethanol. Most other glycerophospholipids (phosphoethanolamines, phosphatidic acids and phosphoglycerols) are less soluble in ethanol, but solutions up to 1–2 mg/mL should be obtainable, using sonication to aid dispersion if necessary. Labeling of cells with fluorescent phospholipids can be enhanced by addition of cyclodextrins during incubation. (J Biol Chem (1999) 274:35359) 5. Alkylpyrene fluorescence lifetimes are up to 110 nanoseconds and are very sensitive to oxygen. 6. Pyrene derivatives exhibit structured spectra. The absorption maximum is usually about 340 nm with a subsidiary peak at about 325 nm. There are also strong absorption peaks below 300 nm. The emission maximum is usually about 376 nm with a subsidiary peak at 396 nm. Excimer emission at about 470 nm may be observed at high concentrations. 7. This product is supplied as a ready-made solution in the solvent indicated under "Soluble." 8. Fluorescence of NBD and its derivatives in water is relatively weak. QY and τ increase and Em decreases in aprotic solvents and other nonpolar environments relative to water. (Biochemistry (1977) 16:5150, Photochem Photobiol (1991) 54:361) PRODUCT LIST 13.3 SPHINGOLIPIDS, STEROIDS, LIPOPOLYSACCHARIDES AND RELATED PROBES Cat. No. Product A12216 Amplex® Red Cholesterol Assay Kit *500 assays* Quantity A12220 Amplex® Red Sphingomyelinase Assay Kit *500 assays* B22650 BODIPY® FL C5-ceramide complexed to BSA 5 mg 1 kit 1 kit B13950 BODIPY® FL C5-ganglioside GM1 25 µg B34401 BODIPY® FL C5-ganglioside GM1 complexed to BSA 1 mg B34402 BODIPY® FL C5-lactosylceramide complexed to BSA D7540 BODIPY® TR ceramide (N-((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)sphingosine) B34400 BODIPY® TR ceramide complexed to BSA 5 mg C12680 cholesteryl BODIPY® 542/563 C11 (cholesteryl 4,4-difluoro-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoate) 1 mg C12681 cholesteryl BODIPY® 576/589 C11 (cholesteryl 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoate) 1 mg C3927MP cholesteryl BODIPY® FL C12 (cholesteryl 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoate) 1 mg D7519 N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)sphingosyl 1-β-D-galactopyranoside (BODIPY® FL C12-galactocerebroside) 25 µg D7711 N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)sphingosyl phosphocholine (BODIPY® FL C12-sphingomyelin) *1 mg/mL in DMSO* 250 µL D3521 N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)sphingosine (BODIPY® FL C5-ceramide) 250 µg 1 mg 250 µg D13951 N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)sphingosyl 1-β-D-lactoside (BODIPY® FL C5-lactosylceramide) 25 µg D3522 N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)sphingosyl phosphocholine (BODIPY® FL C5-sphingomyelin) 250 µg D6562 1,2-dioleoyl-3-(1-pyrenedodecanoyl)-rac-glycerol L23351 lipopolysaccharides from Escherichia coli serotype 055:B5, Alexa Fluor® 488 conjugate 100 µg L23352 lipopolysaccharides from Escherichia coli serotype 055:B5, Alexa Fluor® 568 conjugate 100 µg L23353 lipopolysaccharides from Escherichia coli serotype 055:B5, Alexa Fluor® 594 conjugate 100 µg L23350 lipopolysaccharides from Escherichia coli serotype 055:B5, BODIPY® FL conjugate 100 µg L23356 lipopolysaccharides from Salmonella minnesota, Alexa Fluor® 488 conjugate 100 µg N1154 NBD C6-ceramide (6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosine) 1 mg N22651 NBD C6-ceramide complexed to BSA 5 mg N3524 NBD C6-sphingomyelin (6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosyl phosphocholine) N1148 NBD cholesterol (22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol) P20495 Pro-Q® Emerald 300 Lipopolysaccharide Gel Stain Kit *10 minigels* 1 kit V34403 Vybrant® Alexa Fluor® 488 Lipid Raft Labeling Kit *50 labelings* 1 kit V34404 Vybrant® Alexa Fluor® 555 Lipid Raft Labeling Kit *50 labelings* 1 kit V34405 Vybrant® Alexa Fluor® 594 Lipid Raft Labeling Kit *50 labelings* 1 kit The MolecularProbes® Probes Handbook: Handbook: AAGuide Probes and and Labeling LabelingTechnologies Technologies The Molecular Guideto toFluorescent Fluorescent Probes ™ 574 IMPORTANT NOTICE: The products described in this manual coveredare by covered one or more Limited Use Label License(s). Please refer to thePlease Appendix onto IMPORTANT NOTICE : The products described in thisaremanual by one or more Limited Use Label License(s). refer page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes 1 mg 1 mg 10 mg Chapter 13 — Probes for Lipids and Membranes Section 13.4 Dialkylcarbocyanine and Dialkylaminostyryl Probes 13.4 Dialkylcarbocyanine and Dialkylaminostyryl Probes The dyes in this section are all amphiphilic probes—molecules that comprise a charged fluorophore that localizes the probe at the membrane’s surface and lipophilic aliphatic "tails" that insert into the membrane and thus anchor the probe to the membrane. In addition to labeling model membranes, most of these probes are very useful for cell tracing applications (Section 14.4). Table 14.3 lists all of our lipophilic carbocyanine and aminostyryl tracers and compares their properties and uses. Our FM® dyes, which are also amphiphilic styryl dyes but with less lipophilic character than the dyes in this section, are particularly useful for labeling membranes of live cells and for following synaptosome recycling (Section 16.1). Figure 13.4.3 DiIC12(3) (1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; D383) Dialkylcarbocyanine Probes Carbocyanines are among the most strongly light-absorbing dyes known and have proven to be useful tools in several different areas of research. Carbocyanines with short alkyl tails attached to the imine nitrogens are employed both as membrane-potential sensors (Section 22.3) and as organelle stains for mitochondria and the endoplasmic reticulum (Section 12.2, Section 12.4). Those with longer alkyl tails (≥12 carbons) have an overall lipophilic character that makes them useful for neuronal tracing1 and long-term labeling of cells in culture2,3 (Section 14.4), as well as for noncovalent labeling of lipoproteins (Section 16.1). This section describes the use and properties of dialkylcarbocyanines as general-purpose probes of membrane structure and dynamics. Figure 13.4.4 'DiD'; DiIC18(5) (1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine perchlorate; D307). DiI, DiO, DiD, DiR and Analogs The most widely used carbocyanine membrane probes have been the octadecyl (C18) indocarbocyanines (D282, D3911; Figure 13.4.1) and oxacarbocyanines (D275, Figure 13.4.2) often referred to by the generic acronyms DiI and DiO, or more specifically as DiIC18(3) and DiOC18(3), where the subscript is the number of carbon atoms in each alkyl tail and the bracketed numeral is the number of carbon atoms in the bridge between the indoline or benzoxazole ring systems. We also offer several variations on these basic structures (Section 14.4, Table 14.3): Figure 13.4.5 'DiR'; DiIC18(7) (1,1’-dioctadecyl-3,3,3’,3’-tetramethylindotricarbocyanine iodide; D12731) • DiI and DiO analogs with unsaturated alkyl tails (Δ9-DiI, D3886; FAST DiO™, D3898; FAST DiI™, D3899, D7756) • DiI and DiO analogs with shorter alkyl tails (DiIC12(3), D383; Figure 13.4.3; DiIC16(3), D384; DiOC16(3), D1125) • Long-wavelength light–excitable carbocyanines (DiD, D307, D7757; Figure 13.4.4) • Infrared light–excitable carbocyanine (DiR, D12731; Figure 13.4.5) • Chloromethylbenzamido DiI and sulfonated DiI and DiO derivatives Spectral Properties of Dialkylcarbocyanines The spectral properties of dialkylcarbocyanines are largely independent of the lengths of the alkyl chains, and are instead determined by the heteroatoms in the terminal ring systems and the length of the connecting bridge. The DiICn(3) probes have absorption and fluorescence spectra compatible with rhodamine (TRITC) optical filter sets (Figure 13.4.6), whereas DiOCn(3) analogs can be used with fluorescein (FITC) optical filter sets (Figure 13.4.7). The emission maxima of Figure 13.4.1 'DiI'; DiIC18(3) (1,1'-dioctadecyl-3,3,3',3'tetramethylindocarbocyanine perchlorate; D282). Figure 13.4.2 'DiO'; DiOC18(3) (3,3'-dioctadecyloxacarbocyanine perchlorate; D275). Figure 13.4.6 Absorption and fluorescence emission spectra of DiIC18(3) ("DiI") bound to phospholipid bilayer membranes. Figure 13.4.7 Absorption and fluorescence emission spectra of DiOC18(3) ("DiO") bound to phospholipid bilayer membranes. ™ The Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies TheMolecular Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPORTANT NOTICE:described The products described thiscovered manual are by oneLimited or moreUse Limited Use Label License(s). to the Appendix IMPORTANT NOTICE : The products in this manualinare by covered one or more Label License(s). PleasePlease referrefer to the Appendix on on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes 575 Chapter 13 — Probes for Lipids and Membranes Dil DiD DiR Fluorescence emission DiO 500 600 700 800 900 Wavelength (nm) Figure 13.4.8 Normalized fluorescence emission spectra of DiO (D275), DiI (D282), DiD (D307) and DiR (D12731) bound to phospholipid bilayer membranes. Section 13.4 Dialkylcarbocyanine and Dialkylaminostyryl Probes DiIC18(3) and DiOC18(3) incorporated in dioctadecenoylphosphocholine (dioleoyl PC or DOPC) liposomes (Figure 13.4.8) are similar to those of the dyes in methanol. The very large molar extinction coefficients of carbocyanine fluorophores are their most outstanding spectral property. Their fluorescence quantum yields are only modest—about 0.07 for DiI in methanol and about three-times greater in amphiphilic solvents such as octanol.4,5 Their fluorescence in water is quite weak.6 The excited-state lifetimes of carbocyanine fluorophores in lipid environments are short (~1 nanosecond), which is an advantage for flow cytometry applications because it allows more excitation/de-excitation cycles during flow transit; the overall decay is multi-exponential.7 Dialkylcarbocyanines are also exceptionally photostable.8 The red He-Ne laser–excitable indodicarbocyanines such as DiD (DiIC18(5); D307, D7757) have long-wavelength absorption and red emission (Figure 13.4.8, Figure 13.4.9). Their extinction coefficients are somewhat larger and fluorescence quantum yields much larger than those of carbocyanines such as DiI.5 Moreover, photoexcitation of DiD seems to cause less collateral damage than photoexcitation of DiI in live cells.9 The DiIC18(7) tricarbocyanine probe (DiR, D12731) has excitation and emission in the infrared (Figure 13.4.10), which may make the dye useful as an in vivo tracer for labeled cells and liposomes in live organisms.4,10 Substituted DiI and DiO Derivatives We have synthesized various derivatives of DiI, DiO and DiD. All of these derivatives have octadecyl (C18) tails identical to those of DiI (D282, D3911) and DiO (D275), thereby preserving the excellent membrane retention characteristics of the parent molecules. A variety of substitutions have been made on the indoline or benzoxazole ring systems: Figure 13.4.9 Absorption and fluorescence emission spectra of DiIC18(5) ("DiD") bound to phospholipid bilayer membranes. • Chloromethylbenzamido DiI derivatives (CellTracker™ CM-DiI; C7000, C7001; Figure 13.4.11) • Anionic sulfophenyl derivatives11 of DiI and DiO (SP-DiIC18(3), D7777, Figure 13.4.12; SPDiOC18(3), D7778, Figure 13.4.13) • Sulfonate derivatives of DiI and DiD (DiIC18(3)-DS, D7776, Figure 13.4.14; DiIC18(5)-DS, D12730, Figure 13.4.15) Although these derivatives have primarily been developed to provide improved fixation and labeling in long-term cell tracing applications (Section 14.4), they also offer several features that can potentially be exploited for investigating membrane structure and dynamics. For researchers wishing to carry out comparative evaluations, our Lipophilic Tracer Sampler Kit (L7781) provides 1 mg samples of each of nine different carbocyanine derivatives, including several of the newer substituted derivatives: • DiI (DiIC18(3)) • DiD (DiIC18(5)) • DiR (DiIC18(7)) Figure 13.4.10 Fluorescence excitation and emission spectra of DiIC18(7) ("DiR") bound to phospholipid bilayer membranes. • DiO (DiOC18(3)) • DiA (4-Di-16-ASP) • DiIC18(3)-DS The fluorescence quantum yields of the sulfophenyl and phenyl derivatives (measured in methanol) are generally 2- to 3-fold greater than those of DiI and DiO. In particular, we have found that the sulfophenyl derivatives (SP-DiIC18(3), D7777; SP-DiOC18(3), D7778) bound to phospholipid model membranes have approximately 5-fold higher quantum yields than DiI and DiO. DiIC18(5)DS (D12730) has been used in combination with an NBD-labeled glycerophosphoserine probe in a novel resonance energy transfer assay that detects inner monolayer membrane hemifusion, avoiding erroneous indications of membrane fusion due to lipid mixing and other environmental effects in the outer monolayer.12 The negative charge and greater water solubility of the sulfonated carbocyanines results in modified lateral and transverse distributions of these probes in lipid bilayers relative to those of DiI and DiO. This characteristic has been exploited to identify plasma membrane lipid domains that are responsive to electrical stimulation of outer hair cells in the inner ear.13 Figure 13.4.11 CellTracker™ CM-DiI (C7000). TheMolecular MolecularProbes® Probes Handbook: Handbook: AAGuide and Labeling Labeling Technologies Technologies The GuidetotoFluorescent Fluorescent Probes Probes and ™ 576 • SP-DiIC18(3) • SP-DiOC18(3) • 5,5ʹ-Ph2-DiIC18(3) IMPORTANT NOTICE: The products described in this manual covered one or more Limited Use Label License(s). Please refer to the Appendix IMPORTANT NOTICE : The products described in thisare manual arebycovered by one or more Limited Use Label License(s). Please referonto page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes Chapter 13 — Probes for Lipids and Membranes Section 13.4 Dialkylcarbocyanine and Dialkylaminostyryl Probes DiI and DiO as Probes of Membrane Structure The orientation of DiIC18(3) in membranes has been determined by fluorescence polarization microscopy.14 The long axis of the fluorophore is parallel to the membrane surface, and the two alkyl chains protrude perpendicularly into the lipid interior. There are conflicting reports in the literature regarding the ease of transbilayer migration ("flip-flop") of lipophilic indocarbocyanines.15–17 The lateral partitioning behavior of dialkylindocarbocyanines in membranes has been investigated by fluorescence recovery after photobleaching (FRAP),18 calorimetry,19 lifetime measurements8 and fluorescence resonance energy transfer techniques20 (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2). These studies demonstrate that the probe distribution between coexisting fluid and gel phases depends on the similarity of the alkyl chain lengths of the probe and the lipid. In general, the more dissimilar the lengths, the greater the preference for fluid-phase over gel-phase lipids. For example, the shorter-chain DiIC12(3) has a substantial preference for the fluid phase (~6:1) in DOPC, whereas DiIC18(3) is predominantly distributed in the gel phase21 (~1:10). Consequently, long-chain dialkylcarbocyanines are among the best probes for detecting particularly rigid gel phases. Lipophilic carbocyanines have been used to visualize membrane fusion and cell permeabilization that occurs in response to electric fields,22–24 as well as fusion of liposomes with planar bilayers.25 Membrane fusion can also be measured by fluorescence resonance energy transfer to DiIC18(3) from dansyl- or NBD-labeled phospholipid donors26 or by direct imaging.27 In Langmuir–Blodgett films, excited-state energy transfer from DiIC18(3) to DiIC18(5) is exceptionally efficient because of the favorable orientations of the fluorophores.28 Energy transfer from DiIC18(5) to DiIC18(7) should be similarly effective. Lipophilic carbocyanines have also been used to elicit photosensitized destabilization of liposomes, 29 to sensitize photoaffinity labeling of the viral glycoprotein hemagglutinin, 30 to image membrane domains in lipid monolayers31 and to develop a fiber-optic potassium sensor.32 DiI and DiO as Probes of Membrane Dynamics Despite their reasonably good photostability, dialkylcarbocyanines are widely employed to measure lateral diffusion processes using fluorescence recovery after photobleaching (FRAP) techniques.33–36 Their lateral diffusion coefficients in isolated fluid- and gel-phase bilayers are independent of the carbocyanine alkyl chain length.18 Phase-separated populations of lipophilic carbocyanine dyes can be distinguished by their diffusion rates and can therefore be used to define lateral domains in cell membranes.37,38 Combined lateral diffusion measurements of labeled proteins and lipids have demonstrated that transformed39 and permeabilized40 cells show marked changes in protein diffusion, whereas lipid diffusion rates remain unchanged. In other cases, coupling of lipid and protein mobility has been identified in the form of relatively immobilized lipid domains in yeast plasma membranes41 and around IgE receptor complexes.42 A different photobleaching technique, which depends on the absence of diffusional fluorescence recovery, was employed to determine lipid flow direction in locomoting cells by following the movement of a photobleached stripe of DiIC16(3)43 (D384). H3C The lipophilic aminostyryl probes 4-Di-10-ASP (D291, Figure 13.4.16), DiA (4-Di-16-ASP, D3883; Figure 13.4.17) and FAST DiA™ (D7758, Figure 13.4.18) insert in membranes with their two alkyl CH N CH CH CH3 N (CH2)�� (CH2)�� O3� �O3H CH3 CH3 Figure 13.4.12 1,1’-Dioctadecyl-6,6’-di(4-sulfophenyl)-3,3,3’,3’-tetramethylindocarbocyanine (SP-DiIC18(3), D7777). O CH N CH CH (CH2)�� O3� N (CH2)�� N� CH3 O �O3 CH3 Figure 13.4.13 3,3’-Dioctadecyl-5,5’-di(4-sulfophenyl)oxacarbocyanine, sodium salt (SP-DiOC18(3), D7778). H3C O3� H3C CH3 CH N CH CH CH3 �O3H N (CH2)�� (CH2)�� CH3 CH3 Figure 13.4.14 DiIC18(3)-DS (1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine-5,5’-disulfonic acid; D7776). H3C O3� H3C CH3 (CH N CH)2 CH CH3 �O3H N (CH2)�� (CH2)�� CH3 CH3 Figure 13.4.15 DiIC18(5)-DS (1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine-5,5’disulfonic acid; D12730). CH3N CH CH N�(CH2)�CH3�2 � Figure 13.4.16 4-(4-(Didecylamino)styryl)-N-methylpyridinium iodide (4-Di-10-ASP, D291). Figure 13.4.17 DiA; 4-Di-16-ASP (4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide; D3883). CH3N Dialkylaminostyryl Probes H3C CH3 CH CH N�(CH2)�CH CH CH2 CH CH(CH2)�CH3�2 O3� C� Figure 13.4.18 4-(4-(Dilinoleylamino) styryl)-N-methylpyridinium 4-chlorobenzenesulfonate (FAST DiA™ solid; DiΔ9,12-C18ASP, CBS; D7758). ™ The Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies TheMolecular Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes 577 Chapter 13 — Probes for Lipids and Membranes Section 13.4 Dialkylcarbocyanine and Dialkylaminostyryl Probes Figure 13.4.19 Fluorescence excitation and emission spectra of DiA bound to phospholipid bilayer membranes. Figure 13.4.20 N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM® 1-43, T3163). tails and their fluorophore oriented parallel to the phospholipid acyl chains.44 When these dialkylaminostyryl probes bind to membranes, they exhibit a strong fluorescence enhancement; their fluorescence in water is minimal. The interfacial solvation of the aminostyryl fluorophore causes a large blue shift of the absorption spectrum of the membrane-bound probe.44 For example, the absorption maximum of DiA is 456 nm when incorporated into DOPC liposomes and 490 nm when in methanol. The fluorescence emission maximum of DiA in the membrane environment is 590 nm, which is quite close to that observed for probes with shorter alkyl tails such as 4-Di-10-ASP;44 however, the fluorescence spectrum of DiA is very broad, with appreciable intensity from about 510 nm to 690 nm (Figure 13.4.19). Consequently, DiA can be detected as green, orange or even red fluorescence, depending on the optical filter employed. Like the lipophilic carbocyanines, DiA is commonly used for neuronal membrane tracing (Section 14.4). FAST DiA™ (D7758), the diunsaturated analog of DiA, is intended to facilitate these studies by accelerating dye diffusion within the membrane. The FM® 1-43 (Figure 13.4.20), FM® 1-43FX, FM® 4-64 and FM® 5-95 dyes, which are discussed in detail in Section 16.1, are styryl dyes that also exhibit high Stokes shifts and broad fluorescence emission but have less lipophilic character than the 4-Di-10-ASP and DiA probes. The FM® dyes are commonly used to define the outer membranes of liposomes and live cells and to detect synaptosome recycling. REFERENCES 1. Trends Neurosci (1989) 12:333, 340; 2. Histochemistry (1992) 97:329; 3. Brain Res (2008) 1215:11; 4. J Biomed Opt (2009) 14:054005; 5. Biochemistry (1974) 13:3315; 6. Chem Phys Lipids (2001) 109:175; 7. Biochemistry (1985) 24:5176; 8. J Cell Biol (1985) 100:1309; 9. J Histochem Cytochem (1984) 32:608; 10. J Am Chem Soc (2007) 129:5798; 11. Bioorg Med Chem Lett (1996) 6:1479; 12. Biochim Biophys Acta (2000) 1467:227; 13. J Assoc Res Otolaryngol (2002) 3:289; 14. Biophys J (1979) 26:557; 15. J Cell Biol (1986) 103:807; 16. Biochemistry (1985) 24:582; 17. Nature (1981) 294:718; 18. Biochemistry (1980) 19:6199; 19. Biochim Biophys Acta (1994) 1191:164; 20. Biochim Biophys Acta (2000) 1467:101; 21. Biochim Biophys Acta (1990) 1023:25; 22. Biophys J (1994) 67:427; 23. Biophys J (1993) 65:568; 24. Biochemistry (1990) 29:8337; 25. J Membr Biol (1989) 109:221; 26. Biochim Biophys Acta (1983) 735:243; 27. J Cell Biol (1993) 121:543; 28. Chem Phys Lett (1989) 159:231; 29. FEBS Lett (2000) 467:52; 30. J Biol Chem (1994) 269:14614; 31. Biophys J (1993) 65:1019; 32. Analyst (1990) 115:353; 33. Biochemistry (1977) 16:3836; 34. Biophys J (1998) 75:1131; 35. Biophys J (1995) 68:766; 36. Bioessays (1987) 6:117; 37. Chem Phys Lipids (1994) 73:139; 38. J Cell Biol (1991) 112:1143; 39. Biochim Biophys Acta (1992) 1107:193; 40. J Cell Physiol (1994) 158:7; 41. J Membr Biol (1993) 131:115; 42. J Cell Biol (1994) 125:795; 43. Science (1990) 247:1229; 44. Biophys J (1981) 34:353. DATA TABLE 13.4 DIALKYLCARBOCYANINE AND DIALKYLAMINOSTYRYL PROBES Cat. No. MW Storage Soluble Abs EC Em Solvent Notes C7000 1051.50 F,D,L DMSO, EtOH 553 134,000 570 MeOH C7001 1051.50 F,D,L DMSO, EtOH 553 134,000 570 MeOH D275 881.72 L DMSO, DMF 484 154,000 501 MeOH D282 933.88 L DMSO, EtOH 549 148,000 565 MeOH D291 618.73 L DMSO, EtOH 492 53,000 612 MeOH 1 D307 959.92 L DMSO, EtOH 644 260,000 665 MeOH 2 D383 765.56 L DMSO, EtOH 549 144,000 565 MeOH 3 D384 877.77 L DMSO, EtOH 549 148,000 565 MeOH D1125 825.61 L DMSO, DMF 484 156,000 501 MeOH D3883 787.05 L DMSO, EtOH 491 52,000 613 MeOH 1 D3886 925.49 F,L,AA DMSO, EtOH 549 144,000 564 MeOH 2 D3898 873.65 F,L,AA DMSO, DMF 484 138,000 499 MeOH D3899 925.82 F,L,AA DMSO, EtOH 549 143,000 564 MeOH 2 D3911 933.88 L DMSO, EtOH 549 148,000 565 MeOH D7756 1017.97 F,L,AA DMSO, EtOH 549 148,000 564 MeOH D7757 1052.08 L DMSO, EtOH 644 193,000 663 MeOH D7758 899.80 F,L,AA DMSO, EtOH 492 41,000 612 MeOH 1 D7776 993.54 L DMSO, EtOH 555 144,000 570 MeOH D7777 1145.73 L DMSO, EtOH 556 164,000 573 MeOH D7778 1115.55 L DMSO, EtOH 497 175,000 513 MeOH D12730 1019.58 L DMSO, EtOH 650 247,000 670 MeOH D12731 1013.41 L DMSO, EtOH 748 270,000 780 MeOH For definitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages. Notes 1. Abs and Em of styryl dyes are at shorter wavelengths in membrane environments than in reference solvents such as methanol. The difference is typically 20 nm for absorption and 80 nm for emission, but varies considerably from one dye to another. Styryl dyes are generally nonfluorescent in water. 2. This product is intrinsically a liquid or an oil at room temperature. 3. This product is intrinsically a sticky gum at room temperature. The MolecularProbes® Probes Handbook: Handbook: AAGuide Probesand andLabeling LabelingTechnologies Technologies The Molecular Guide to to Fluorescent Fluorescent Probes ™ 578 IMPORTANT NOTICE: The products described in this manual coveredare by covered one or more Limited Use Label License(s). Please refer to thePlease Appendix onto IMPORTANT NOTICE : The products described in thisaremanual by one or more Limited Use Label License(s). refer page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes Chapter 13 — Probes for Lipids and Membranes Section 13.5 Other Nonpolar and Amphiphilic Probes PRODUCT LIST 13.4 DIALKYLCARBOCYANINE AND DIALKYLAMINOSTYRYL PROBES Cat. No. Product C7001 CellTracker™ CM-DiI Quantity C7000 CellTracker™ CM-DiI *special packaging* D291 4-(4-(didecylamino)styryl)-N-methylpyridinium iodide (4-Di-10-ASP) D383 1,1’-didodecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiIC12(3)) D3883 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA; 4-Di-16-ASP) D1125 3,3’-dihexadecyloxacarbocyanine perchlorate (DiOC16(3)) D384 1,1’-dihexadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiIC16(3)) D7758 4-(4-(dilinoleylamino)styryl)-N-methylpyridinium 4-chlorobenzenesulfonate (FAST DiA™ solid; DiΔ9,12-C18ASP, CBS) D3898 3,3’-dilinoleyloxacarbocyanine perchlorate (FAST DiO™ solid; DiOΔ9,12-C18(3), ClO4) 5 mg D7756 1,1’-dilinoleyl-3,3,3’,3’-tetramethylindocarbocyanine, 4-chlorobenzenesulfonate (FAST DiI™ solid; DiIΔ9,12-C18(3), CBS) 5 mg D3899 1,1’-dilinoleyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (FAST DiI™ oil; DiIΔ9,12-C18(3), ClO4) 5 mg D7778 3,3’-dioctadecyl-5,5’-di(4-sulfophenyl)oxacarbocyanine, sodium salt (SP-DiOC18(3)) 5 mg D7777 1,1’-dioctadecyl-6,6’-di(4-sulfophenyl)-3,3,3’,3’-tetramethylindocarbocyanine (SP-DiIC18(3)) D275 3,3’-dioctadecyloxacarbocyanine perchlorate (‘DiO’; DiOC18(3)) D7776 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine-5,5’-disulfonic acid (DiIC18(3)-DS) D282 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (‘DiI’; DiIC18(3)) 1 mg 20 x 50 µg 25 mg 100 mg 25 mg 25 mg 100 mg 5 mg 5 mg 100 mg 5 mg 100 mg D3911 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate *crystalline* (‘DiI’; DiIC18(3)) 25 mg D7757 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (‘DiD’ solid; DiIC18(5) solid) 10 mg D12730 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine-5,5’-disulfonic acid (DiIC18(5)-DS) 5 mg D307 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine perchlorate (‘DiD’ oil; DiIC18(5) oil) 25 mg D12731 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindotricarbocyanine iodide (‘DiR’; DiIC18(7)) 10 mg D3886 1,1’-dioleyl-3,3,3’,3’-tetramethylindocarbocyanine methanesulfonate (Δ9-DiI) 25 mg L7781 Lipophilic Tracer Sampler Kit 1 kit 13.5 Other Nonpolar and Amphiphilic Probes Amphiphilic Rhodamine, Fluorescein and Coumarin Derivatives Each of our amphiphilic probes comprises a moderately polar fluorescent dye with a lipophilic "tail." When used to stain membranes, including liposomes, the lipophilic portion of the probe tends to insert in the membrane and the polar fluorophore resides on the surface, where it senses the membrane’s surface environment and the surrounding medium.1 Our lipophilic carbocyanines and styryl dyes (Section 13.4) are also amphiphilic molecules with a similar binding mode. This section includes the classic membrane probes DPH, TMA-DPH, ANS, bis-ANS, TNS, prodan, laurdan and nile red, and also some lipophilic BODIPY® and Dapoxyl® dyes developed in our laboratories. Although they bear little resemblance to natural products, these probes tend to localize within cell membranes or liposomes or at their aqueous interfaces, where they are often used to report on characteristics of their local environment, such as viscosity, polarity and lipid order. Figure 13.5.1 Octadecyl rhodamine B chloride (O246). Octadecyl Rhodamine B The relief of the fluorescence self-quenching of octadecyl rhodamine B (O246, Figure 13.5.1) can be used to monitor membrane fusion 2–7—one of several experimental approaches developed for this application (Lipid-Mixing Assays of Membrane Fusion—Note 13.1). Octadecyl rhodamine B has been reported to undergo a potential-dependent "flip-flop" from one monolayer of a fluid-state phospholipid bilayer membrane to the other, with partial relief of its fluorescence quenching.8,9 Investigators have used octadecyl rhodamine B in conjunction with video microscopy 10–12 or digital imaging techniques 13 to monitor viral fusion processes. Membrane fusion can ™ The Probes Handbook: A Guide to Fluorescent Probes andand Labeling Technologies TheMolecular Molecular Probes® Handbook: A Guide to Fluorescent Probes Labeling Technologies IMPORTANT NOTICE: The products described thiscovered manual by areone covered by one or more Limited Label License(s). refer the Appendix IMPORTANT NOTICE : The products described in this manualinare or more Limited Use LabelUse License(s). PleasePlease refer to thetoAppendix on on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes 579 Chapter 13 — Probes for Lipids and Membranes HO O O C OH O NH C O (CH2)��CH3 Figure 13.5.2 5-Hexadecanoylaminofluorescein (H110). Section 13.5 Other Nonpolar and Amphiphilic Probes also be followed by monitoring fluorescence resonance energy transfer to octadecyl rhodamine B from an acylaminofluorescein donor such as 5-hexadecanoylaminofluorescein 5,7,14,15 (H110, Figure 13.5.2). Fluorescence resonance energy transfer from fluorescein or dansyl labels to octadecyl rhodamine B has been used for structural studies of the blood coagulation factor IXa, EGF receptor and receptor-bound IgE.16–18 Octadecyl rhodamine B has also been used to stain kinesin-generated membrane tubules,19 to characterize detergent micelles,20 to assay for lysosomal degradation of lipoproteins 21 and to investigate the influence of proteins on lipid dynamics using time-resolved fluorescence anisotropy.22 Amphiphilic Fluoresceins HO O O C O(CH2)��CH3 O Figure 13.5.3 Fluorescein octadecyl ester (F3857). The amphiphilic fluorescein probes bind to membranes with the fluorophore at the aqueous interface and the alkyl tail protruding into the lipid interior. 5-Dodecanoylaminofluorescein (D109) is the hydrolysis product of our ImaGene Green™ C12-FDG β-galactosidase substrate (D2893, Section 10.2). We also offer the homologous membrane probe 5-hexadecanoylaminofluorescein 1,15,23 (H110, Figure 13.5.2) and the octadecyl ester of fluorescein 24,25 (F3857, Figure 13.5.3). Amphiphilic fluorescein probes are commonly used for fluorescence recovery after photobleaching (FRAP) measurements of lipid lateral diffusion.26 Some researchers have reported that 5-hexadecanoylaminofluorescein stays predominantly in the outer membrane leaflet of epithelia and does not pass through tight junctions, whereas the dodecanoyl derivative can "flip-flop" to the inner leaflet at 20°C (but not at <10°C) and may also pass through tight junctions.27,28 More recent studies have indicated that the lack of tight junction penetration of 5-hexadecanoylaminofluorescein is due to probe aggregation rather than a significant difference in its transport properties.29 Amphiphilic Coumarin Figure 13.5.4 4-Heptadecyl-7-hydroxycoumarin (H22730). Figure 13.5.5 DPH (1,6-diphenyl-1,3,5-hexatriene; D202). 4-Heptadecyl-7-hydroxycoumarin (H22730, Figure 13.5.4) is an alkyl derivative of the pH-sensitive blue-fluorescent 7-hydroxycoumarin (umbelliferone) fluorophore. As with other amphiphilic coumarins, 30 4-heptadecyl-7-hydroxycoumarin is primarily useful as a probe of membrane surfaces. Deprotonation of the 7-hydroxyl group is expected to be strongly dependent on membrane-surface electrostatic potential. The pKa of 4-heptadecyl-7-hydroxycoumarin varies from 6.35 in the cationic detergent CTAB to 11.15 in the anionic detergent sodium dodecyl sulfate (SDS), as measured by its fluorescence response.31 However, its pKa in lipid assemblies is strongly dependent on the ionic composition of the membrane surface, 31,32 making it a sensitive probe of membrane-surface electrostatic potential.33 4-Heptadecyl-7-hydroxycoumarin has been used to measure pH differences at membrane interfaces in isolated plasma membranes of normal and multidrug-resistant murine leukemia cells.34,35 4-Heptadecyl-7-hydroxycoumarin has also been employed as a structural probe for the head-group region of phospholipid bilayers. 36 DPH and DPH Derivatives Diphenylhexatriene (DPH) Figure 13.5.6 TMA-DPH (1-(4-trimethylammoniumphenyl)6-phenyl-1,3,5-hexatriene p-toluenesulfonate; T204). Figure 13.5.7 4,4-Difluoro-1,3,5,7,8-pentamethyl-4-bora3a,4a-diaza-s-indacene (BODIPY® 493/503, D3922). 1,6-Diphenyl-1,3,5-hexatriene (DPH, D202; Figure 13.5.5) continues to be a popular fluorescent probe of membrane interiors. We also offer the cationic DPH derivative TMA-DPH, as well as the phospholipid analog (D476, Section 13.2). The orientation of DPH within lipid bilayers is loosely constrained. It is generally assumed to be oriented parallel to the lipid acyl chain axis, but it can also reside in the center of the lipid bilayer parallel to the surface, as demonstrated by time-resolved fluorescence anisotropy and polarized fluorescence measurements of oriented samples.37–40 DPH shows no partition preference between coexisting gel- and fluid-phase phospholipids.41 Intercalation of DPH and its derivatives into membranes is accompanied by strong enhancement of their fluorescence; their fluorescence is practically negligible in water. The fluorescence decay of DPH in lipid bilayers is complex.42–44 Fluorescence decay data are often analyzed in terms of continuous lifetime distributions,45–48 which are in turn interpreted as being indicative of lipid environment heterogeneity. DPH and its derivatives are cylindrically shaped molecules with absorption and fluorescence emission transition dipoles aligned approximately parallel to their long molecular The MolecularProbes® Probes Handbook: Handbook: A Probesand andLabeling LabelingTechnologies Technologies The Molecular A Guide Guide to to Fluorescent Fluorescent Probes ™ 580 IMPORTANT NOTICE: The products described in this manual aremanual coveredare by one or more Use Label License(s). Please refer to thePlease Appendix on to IMPORTANT NOTICE : The products described in this covered by Limited one or more Limited Use Label License(s). refer page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes Chapter 13 — Probes for Lipids and Membranes Section 13.5 Other Nonpolar and Amphiphilic Probes axis. Consequently, their fluorescence polarization is high in the absence of rotational motion and is very sensitive to reorientation of the long axis resulting from interactions with surrounding lipids. These properties have led to their extensive use for membrane fluidity measurements.49 The exact physical interpretation of these measurements has some contentious aspects. For instance, the probes are largely sensitive to only the angular reorientation of lipid acyl chains—a motion that does not necessarily correlate with other dynamic processes such as lateral diffusion.50 Reviews on this subject 39,49,51,52 should be consulted for further discussion. Time-resolved fluorescence polarization measurements of lipid order are more physically rigorous because they allow the angular range of acyl chain reorientation ("lipid order") to be resolved from its rate, and considerable research has been devoted to the interpretation of these measurements.37,45,53,54 TMA-DPH Designed to improve the localization of DPH in the membrane, TMA-DPH (T204, Figure 13.5.6) contains a cationic trimethylammonium substituent that acts as a surface anchor.55–57 Like DPH, this derivative readily partitions from aqueous dispersions into membranes and other lipid assemblies, accompanied by strong fluorescence enhancement. The lipid–water partition coefficient (Kp) for TMA-DPH (Kp = 2.4 × 105) is lower than for DPH (Kp = 1.3 × 106), reflecting the increased water solubility caused by the polar substituents.58 The fluorescence decay lifetime of TMA-DPH is more sensitive to changes in lipid composition and temperature than is the fluorescence decay lifetime of DPH.59–61 Staining of cell membranes by TMA-DPH is much more rapid than staining by DPH; however, the duration of plasma membrane surface staining by TMA-DPH before internalization into the cytoplasm is quite prolonged.62,63 As a consequence, TMA-DPH introduced into Madin–Darby canine kidney (MDCK) cell plasma membranes does not diffuse through tight junctions and remains in the apical domain, whereas the anionic DPH propionic acid accumulates rapidly in intracellular membranes.64 TMA-DPH residing in the plasma membrane can be extracted by washing with medium, thus providing a method for isolating internalized probe and monitoring endocytosis 65 (Section 16.1). Furthermore, because TMA-DPH is virtually nonfluorescent in water and binds in proportion to the available membrane surface,66 its fluorescence intensity is sensitive to increases in plasma membrane surface area resulting from exocytosis.65,67,68 TMA-DPH fluorescence polarization measurements can be combined with video microscopy to provide spatially resolved images of phospholipid order in large liposomes and single cells.69–72 Information regarding lipid order heterogeneity among cell populations can be obtained in a similar way using flow cytometry.73–75 Nonpolar BODIPY® Probes for oils and other nonpolar liquids. In addition, their photostability is generally high; this, together with other favorable characteristics (very low triplet–triplet absorption), make the BODIPY® 493/503 and BODIPY® 505/515 fluorophores excellent choices for flashlamp-pumped laser dyes.78,79 Staining with the BODIPY® 493/503 dye (D3922, Figure 13.5.7) has been shown by flow cytometry to be more specific for cellular lipid droplets than staining with nile red 80 (N1142). The low molecular weight of the BODIPY® 493/503 dye (262 daltons) results in the probe having a relatively fast diffusion rate in membranes.81 The BODIPY® 493/503 dye has also been used to detect neutral compounds in a microchip channel separation device.82 BODIPY® 505/515 (D3921, Figure 13.5.8) rapidly permeates cell membranes of live zebrafish embryos,83,84 selectively staining cytoplasmic yolk platelets. This staining provides dramatic contrast enhancement of cytoplasm relative to nucleoplasm and interstitial spaces, allowing individual cell boundaries and cell nuclei to be imaged clearly with a confocal laser-scanning microscope (Figure 13.5.9). The very long–wavelength BODIPY® 665/676 dye (B3932, Figure 13.5.10) has fluorescence that is not visible to the human eye; however, it has found use as a probe for reactive oxygen species 85 (Section 18.2). CH3 H3C N F H3C B N F CH3 Figure 13.5.8 4,4-Difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY® 505/515, D3921). Figure 13.5.9 Dorsal view of the midbrain/hindbrain region of a 15-somite stage zebrafish embryo labeled with BODIPY® 505/515 (D3921). BODIPY® 505/515 localizes in lipidic yolk platelets, producing selective cytoplasmic staining. This pseudocolored confocal image was obtained using a Bio-Rad® MRC-600 microscope. Image contributed by Mark Cooper, University of Washington. BODIPY® Fluorophores BODIPY® fluorophore derivatives offer an unusual combination of nonpolar structure (Figure 13.5.7) and long-wavelength absorption and fluorescence.76 BODIPY® dyes have small fluorescence Stokes shifts, extinction coefficients that are typically greater than 80,000 cm–1M–1 and high fluorescence quantum yields that are not diminished in water.77 These dyes have applications as stains for neutral lipids and as tracers N CH CH CH CH F B N F CH CH CH CH Figure 13.5.10 (E,E)-3,5-bis-(4-phenyl-1,3-butadienyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY® 665/676, B3932). ™ The Handbook: A Guide to Fluorescent Probes and Labeling Technologies TheMolecular MolecularProbes Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPORTANT NOTICE:described The products described this covered manual are by oneLimited or moreUse Limited UseLicense(s). Label License(s). Please to the Appendix IMPORTANT NOTICE : The products in this manualin are bycovered one or more Label Please referrefer to the Appendix on on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes 581 Chapter 13 — Probes for Lipids and Membranes Section 13.5 Other Nonpolar and Amphiphilic Probes BODIPY® FL C5-Ceramide BODIPY® FL C5-ceramide (D3521, B22650; Section 13.3) stains the plasma membrane, Golgi apparatus and cytoplasmic particles within the superficial enveloping layer (EVL) of embryos. Once the fluorescent lipid percolates through the EVL epithelium, however, it remains localized within the interstitial fluid of the embryo and freely diffuses between cells (Figure 13.5.11). Vital staining with BODIPY® FL C5-ceramide thus allows hundreds of cells to be imaged en masse during morphogenetic movements.83,86 CellTrace™ BODIPY® TR Methyl Ester Figure 13.5.11 Cells in the notochord rudiment of a zebrafish embryo undergoing mediolateral intercalation to lengthen the forming notochord. BODIPY® FL C5-ceramide (D3521) localizes in the interstitial fluid of the zebrafish embryo and freely diffuses between cells, illuminating cell boundaries. This confocal image was obtained using a Bio-Rad® MRC-600 microscope. Image contributed by Mark Cooper, University of Washington. N F B N F � O OCH2 C OCH3 Figure 13.5.12 CellTrace™ BODIPY® TR methyl ester (C34556). CellTrace™ BODIPY® TR methyl ester Absorption Fluorescence emission EGFP 300 350 400 450 500 550 600 650 700 750 Wavelength (nm) Figure 13.5.13 Normalized absorption (—) and fluorescence emission (– – –) spectra of enhanced Green Fluorescent Protein (EGFP) and CellTrace™ BODIPY® TR methyl ester (C34556). Many research and biotechnological applications require detailed three- and four-dimensional visualization of embryonic cells labeled with Green Fluorescent Protein (GFP) within their native tissue environments. Fluorescent counterstains that label all the cells in a living embryo provide a histological context for the GFP-expressing cells in the specimen. The red-fluorescent CellTrace™ BODIPY® TR methyl ester (C34556, Figure 13.5.12) is an excellent counterstain for cells and tissues that are expressing GFP.87 This dye readily permeates cell membranes and selectively stains mitochondria and endomembranous organelles such as endoplasmic reticulum and the Golgi apparatus, but does not appear to localize in the plasma membrane. These localization properties make the dye an ideal vital stain that can be used to reveal: (1) the location and shapes of cell nuclei, (2) the shapes of cells within embryonic tissues and (3) the boundaries of organ-forming tissues within the whole embryo.87 Furthermore, CellTrace™ BODIPY® TR methyl ester staining is retained after formaldehyde fixation and permeabilization with Triton X-100, and the dye does not appear to produce any teratogenic effects on embryonic development. The emission spectra of enhanced GFP (EGFP) and CellTrace™ BODIPY® TR methyl ester are well separated, with peaks at 508 nm and 625 nm, respectively (Figure 13.5.13), allowing simultaneous dual-channel confocal imaging without significant overspill of GFP fluorescence into the CellTrace™ BODIPY® TR methyl ester detection channel. The Image-iT® LIVE Intracellular Membrane and Nuclear Labeling Kit (I34407, Section 14.4) provides the red-fluorescent CellTrace™ BODIPY® TR methyl ester along with the blue-fluorescent Hoechst 33342 dye for highly selective staining of the intracellular membranes and nuclei, respectively, of live or fixed cells or tissues (Figure 13.5.14). These two fluorescent stains were especially chosen for their compatibility with live GFP-expressing cells, and they can be combined into one staining solution to save labeling time and wash steps while still providing optimal staining. Pyrene, Nile Red and Bimane Probes Nonpolar Pyrene Probe 1,3-Bis-(1-pyrene)propane (B311, Figure 13.5.15) has two pyrene moieties linked by a threecarbon alkylene spacer. This probe is somewhat analogous to the bis-pyrenyl phospholipids (Section 13.2) in that excimer formation (and, consequently, the fluorescence emission wavelength) is controlled by intramolecular rather than bimolecular interactions. Thus, this probe is highly sensitive to constraints imposed by its environment, and can therefore be used as a viscosity sensor for interior regions of lipoproteins, membranes, micelles, liquid crystals and synthetic polymers.88 Because excimer formation results in a spectral shift (Figure 13.5.16), the probe may be useful for ratio imaging of molecular mobility.89 However, pyrene fatty acids (Section 13.2) appear to be preferable for this purpose because the uptake of 1,3-bis-(1-pyrene)propane by cells is limited. Nile Red Figure 13.5.14 Live HeLa cells were transfected using pShooter™ vector pCMV/myc/mito/GFP and Lipofectamine® 2000 transfection reagent and stained with the reagents in the Image-iT® LIVE Intracellular Membrane and Nuclear Labeling Kit (I34407). Intracellular membranes were stained with CellTrace™ BODIPY® TR methyl ester, and nuclei were stained with Hoechst 33342. Cells were visualized using epifluorescence microscopy. The phenoxazine dye nile red (N1142, Figure 13.5.17) is used to localize and quantitate lipids, particularly neutral lipid droplets within cells.80,90–92 It is selective for neutral lipids such as cholesteryl esters 93,94 (and also, therefore, for lipoproteins) and is suitable for staining lysosomal phospholipid inclusions.95 Nile red is almost nonfluorescent in water and other polar solvents but undergoes fluorescence enhancement and large absorption and emission blue shifts in nonpolar environments.96,97 Its fluorescence enhancement upon binding to proteins is weaker than that produced by its association with lipids 97 (Figure 13.5.18). Ligand-binding studies on tubulin and tryptophan synthase 98 have exploited the environmental sensitivity of nile red’s fluorescence. Nile red has also been used to detect sphingolipids on thin-layer chromatograms 99 and to stain proteins after SDS-polyacrylamide gel electrophoresis.100 TheMolecular MolecularProbes® Probes Handbook: Handbook: AAGuide and Labeling Labeling Technologies Technologies The GuidetotoFluorescent Fluorescent Probes Probes and ™ 582 IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes Chapter 13 — Probes for Lipids and Membranes Section 13.5 Other Nonpolar and Amphiphilic Probes Bimane Azide (CH2)3 Bimane azide (B30600, Figure 13.5.19) is a small blue-fluorescent photoreactive alkyl azide (excitation/emission maxima ~375/458 nm) for photoaffinity labeling of proteins, potentially including membrane proteins from within the cell membrane. This reactive fluorophore’s small size may reduce the likelihood that the label will interfere with the function of the biomolecule, an important advantage for site-selective probes. Figure 13.5.15 1,3-Bis-(1-pyrenyl)propane (B311). Steatosis, the intracellular accumulation of neutral lipids as lipid droplets or globules, is often triggered by drugs that affect the metabolism of fatty acids or neutral lipids. LipidTOX™ neutral lipid stains were developed to characterize the effects of drugs and other compounds on lipid metabolism in mammalian cell lines. LipidTOX™ neutral lipid stains have an extremely high affinity for neutral lipid droplets. These reagents are added after cell fixation and do not require subsequent wash steps after incubation with the sample. Key advantages of this series of neutral lipid stains over conventional stains such as nile red include their ready-to-use formulations, their flexibility for multiplexing protocols and their compatibility with LipidTOX™ phospholipid stains (H34350, H34351; Section 13.2). LipidTOX™ neutral lipid stains are available with green, red and deep red fluorescence emission: • HCS LipidTOX™ Green neutral lipid stain (H34475), with excitation/emission maxima ~495/505 nm (Figure 13.5.20) • HCS LipidTOX™ Red neutral lipid stain (H34476), with excitation/emission maxima ~577/609 nm • HCS LipidTOX™ Deep Red neutral lipid stain (H34477), with excitation/emission maxima ~637/655 nm A Ex = 370 nm B DOPC 1 2 3 4 350 400 450 450 500 550 Wavelength (nm) 600 550 600 Figure 13.5.16 Excimer formation by pyrene in ethanol. Spectra are normalized to the 371.5 nm peak of the monomer. All spectra are essentially identical below 400 nm after normalization. Spectra are as follows: 1) 2 mM pyrene, purged with argon to remove oxygen; 2) 2 mM pyrene, airequilibrated; 3) 0.5 mM pyrene (argon-purged); and 4) 2 µM pyrene (argon-purged). The monomer-to-excimer ratio (371.5 nm/470 nm) is dependent on both pyrene concentration and the excited-state lifetime, which is variable because of quenching by oxygen. Ex = 540 nm BSA Figure 13.5.17 Nile red (N1142). O 400 500 Wavelength (nm) DOPC Fluorescence emission Fluorescence emission BSA Fluorescence emission LipidTOX™ Neutral Lipid Stains 550 600 650 700 750 CH3 Wavelength (nm) Figure 13.5.18 Fluorescence emission spectra of A) 1,8-ANS (A47) and B) nile red (N1142) bound to protein and phospholipid vesicles. Samples comprised 1 µM dye added to 20 µM bovine serum albumin (BSA) or 100 µM dioctadecenoylglycerophosphocholine (DOPC). CH3 O N N CH3 CH2N N N Figure 13.5.19 Bimane azide (B30600). Figure 13.5.20 FABP4 antibody labeling in adipocytes. Adipocytes differentiated from 3T3-L1 mouse fibroblasts were fixed with formaldehyde and permeabilized with saponin before labeling with rabbit anti–fatty acid binding protein (FABP4) IgG (red). These cells were then stained with LipidTOX™ Green neutral lipid stain (H34475, green), counterstained with DAPI (D1306, D21490; blue) and mounted in ProLong® Gold antifade reagent (P36930). ™ The Handbook: A Guide to Fluorescent Probes and Labeling Technologies TheMolecular MolecularProbes Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPORTANT NOTICE: described The products described in this oneLimited or more Use Limited UseLicense(s). Label License(s). Please to the Appendix IMPORTANT NOTICE : The products in this manual are manual coveredare bycovered one or by more Label Please referrefer to the Appendix onon page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes 583 Chapter 13 — Probes for Lipids and Membranes Figure 13.5.21 Prodan (6-propionyl-2-dimethylaminonaphthalene; P248). Figure 13.5.22 Normalized emission spectra of prodan (P248) excited at 345 nm in 1) cyclohexane, 2) dimethylformamide, 3) ethanol and 4) water. Figure 13.5.23 6-Dodecanoyl-2-dimethylaminonaphthalene (laurdan, D250). Figure 13.5.24 Absorption and fluorescence emission spectra of Dapoxyl® (2-aminoethyl)sulfonamide in methanol. Figure 13.5.25 Dapoxyl® sulfonic acid, sodium salt (D12800). Section 13.5 Other Nonpolar and Amphiphilic Probes These HCS LipidTOX™ neutral lipid stains have been used to image intracellular lipid accumulation in rat cortical neurons, COS-7 cells and hepatitis C virus (HCV)–infected FT3-7 human hepatoma cells.101–103 HCS LipidTOX™ Red neutral lipid stain was used to detect RNAi knockdown of acyl-coenzyme A:cholesterol acyl transferase, isoform 1 (ACAT-1), an endoplasmic reticulum enzyme that regulates the equilibrium between free cholesterol and cholesteryl esters in cells.104 LipidTOX™ Green neutral lipid stain is also a component of the HCS LipidTOX™ Phospholipidosis and Steatosis Detection Kit (H34157, H34158; Section 13.2), which provides a complete set of reagents for performing high-content screening (HCS) assays to detect and distinguish the intracellular accumulation of phospholipids (phospholipidosis) and of neutral lipids (steatosis) in mammalian cell lines after exposure to test compounds. In addition, HCS LipidTOX™ neutral lipid stains can be used to monitor the formation and differentiation of adipocytes, a process called adipogenesis. Adipogenesis is of acute interest to the biomedical and drug discovery community as it plays an important role in diseases such as obesity, diabetes and atherosclerosis. HCS LipidTOX™ neutral lipid stains are designed for fixed–end point workflows in which formaldehyde-fixed cells in microplates are processed, imaged and analyzed. These stains can easily be detected with fluorescence microscopes or HCS readers equipped with standard filter sets. Membrane Probes with Environment-Sensitive Spectral Shifts Prodan and Laurdan Prodan (P248, Figure 13.5.21), introduced by Weber and Farris in 1979, has both electrondonor and electron-acceptor substituent, resulting in a large excited-state dipole moment and extensive solvent polarity–dependent fluorescence shifts 105 (Figure 13.5.22). Several variants of the original probe have since been prepared, including the lipophilic derivative laurdan (D250, Figure 13.5.23) and thiol-reactive derivatives acrylodan and badan (A433, B6057; Section 2.3), which can be used to confer the environment-sensitive properties of this fluorophore on bioconjugates. When prodan or its derivatives are incorporated into membranes, their fluorescence spectra are sensitive to the physical state of the surrounding phospholipids.106 In membranes, prodan appears to localize at the surface,107 although Fourier transform infrared (FTIR) measurements indicate some degree of penetration into the lipid interior.108 Excited-state relaxation of prodan is sensitive to the nature of the linkage (ester or ether) between phospholipid hydrocarbon tails and the glycerol backbone.109 In contrast, laurdan’s excited-state relaxation is independent of headgroup type, and is instead determined by water penetration into the lipid bilayer.110,111 Two-photon infrared excitation techniques have been successfully applied to both prodan and laurdan, although both probes nominally require ultraviolet excitation 112–115 (~360 nm). Much experimental work using these probes has sought to characterize coexisting lipid domains based on their distinctive fluorescence spectra,113,116–120 an approach that is intrinsically amenable to dual-wavelength ratio measurements.111,121 Other applications include detecting nonbilayer lipid phases,122,123 mapping changes in membrane structure induced by cholesterol and alcohols 124–127 and assessing the polarity of lipid/water interfaces.128,129 Like ANS, prodan is also useful as a noncovalently interacting probe for proteins.130–133 Dapoxyl® Derivative We have developed a variety of probes based on our Dapoxyl® fluorophore.134 Dapoxyl® sulfonamide derivatives exhibit UV absorption with maxima near 370 nm, extinction coefficients >24,000 cm–1M–1 and Stokes shifts in excess of 200 nm (Figure 13.5.24). Dapoxyl® sulfonic acid (D12800, Figure 13.5.25) is an amphiphilic Dapoxyl® derivative with generally similar properties and applications to anilinonaphthalene sulfonate (ANS) (Monitoring Protein-Folding Processes with Environment-Sensitive Dyes—Note 9.1). Both ANS and Dapoxyl® sulfonic acid have been used in a drug-discovery assay based on the detection of protein thermal denaturation shifts.135 Reactive versions of the Dapoxyl® fluorophore are described in Section 1.7 and Section 3.4. Anilinonaphthalenesulfonate (ANS) and Related Derivatives The use of anilinonaphthalene sulfonates (ANS) as fluorescent probes dates back to the pioneering work of Weber in the 1950s, and this class of probes remains valuable for studying both membrane The MolecularProbes® Probes Handbook: Handbook: AA Guide Probesand andLabeling LabelingTechnologies Technologies The Molecular Guide to to Fluorescent Fluorescent Probes ™ 584 IMPORTANT NOTICE: The products described in this manual coveredare by one or more Limited Use Label License(s). Please refer to thePlease Appendix onto IMPORTANT NOTICE : The products described in thisaremanual covered by one or more Limited Use Label License(s). refer page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes Chapter 13 — Probes for Lipids and Membranes Section 13.5 Other Nonpolar and Amphiphilic Probes surfaces and proteins. Slavik’s 1982 review of its properties is recommended reading, especially for the extensive compilation of spectral data.136 The primary member of this class, 1,8-ANS (A47, Figure 13.5.26), and its analogs 2,6-ANS (A50) and 2,6-TNS (T53) are all essentially nonfluorescent in water, only becoming appreciably fluorescent when bound to membranes (quantum yields ~0.25) or proteins (quantum yields ~0.7)136–138 (Figure 13.5.18). This property makes them sensitive indicators of protein folding, conformational changes 139–142 and other processes that modify the exposure of the probe to water (Monitoring Protein-Folding Processes with Environment-Sensitive Dyes—Note 9.1). Fluorescence of 2,6-ANS is also enhanced by cyclodextrins, permitting a sensitive method for separating and analyzing cyclodextrins with capillary electrophoresis.143 Figure 13.5.26 1,8-ANS (1-anilinonaphthalene-8-sulfonic acid, A47). Bis-ANS Bis-ANS (B153, Figure 13.5.27) is superior to 1,8-ANS as a probe for nonpolar cavities in proteins, often binding with an affinity that is orders-of-magnitude higher.144–147 Bis-ANS has particularly high affinity for nucleotide-binding sites of some proteins.148–150 It is also useful as a structural probe for tubulin 151,152 and as an inhibitor of microtubule assembly.153–155 Covalent photoincorporation of bis-ANS into proteins has been reported.156 Figure 13.5.27 bis-ANS (4,4’-dianilino-1,1’-binaphthyl-5,5’disulfonic acid, dipotassium salt; B153). DCVJ The styrene derivative DCVJ (D3923, Figure 13.5.28) is a sensitive indicator of tubulin assembly and actin polymerization.157,158 The fluorescence quantum yield of DCVJ is strongly dependent on environmental rigidity, resulting in large fluorescence increases when the dye binds to antibodies 159 and when it is compressed in synthetic polymers or phospholipid membrane interiors.160,161 DCVJ has been used for microviscosity measurements of phospholipid bilayers.161 CH C(CN)2 N Figure 13.5.28 DCVJ (4-(dicyanovinyl)julolidine, D3923). REFERENCES 1. Biochim Biophys Acta (1998) 1374:63; 2. Biophys J (1999) 77:943; 3. Photochem Photobiol (1994) 60:563; 4. Biophys Chem (1989) 34:283; 5. Chem Phys Lipids (2002) 116:39; 6. Biochemistry (1984) 23:5675; 7. J Cell Sci (1977) 28:167; 8. Biophys J (1996) 71:2680; 9. Biochim Biophys Acta (1995) 1237:121; 10. Biophys J (1992) 63:710; 11. J Gen Physiol (1991) 97:1101; 12. Proc Natl Acad Sci U S A (1990) 87:1850; 13. FEBS Lett (1989) 250:487; 14. 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Biophys J (1991) 59:466; 44. Biophys J (1989) 56:723; 45. Biophys Chem (1994) 48:337; 46. Biochim Biophys Acta (1992) 1104:273; 47. Biochemistry (1990) 29:3248; 48. Chem Phys Lipids (1989) 50:1; 49. Chem Phys Lipids (1993) 64:99; 50. Biochim Biophys Acta (1981) 649:471; 51. Chem Phys Lipids (1993) 64:117; 52. Biochim Biophys Acta (1986) 854:38; 53. Chem Phys (1994) 185:393; 54. Chem Phys Lett (1993) 216:559; 55. Biochemistry (1998) 37:8180; 56. Biochemistry (1988) 27:7723; 57. Biochemistry (1981) 20:7333; 58. Biochem Biophys Res Commun (1991) 181:166; 59. Chem Phys Lipids (1990) 55:29; 60. Biochemistry (1987) 26:5121; 61. Biochemistry (1987) 26:5113; 62. Biochim Biophys Acta (1985) 845:60; 63. Cell Biophys (1983) 5:129; 64. Am J Physiol (1988) 255:F22; 65. Biochim Biophys Acta (1995); 66. Biochemistry (1986) 25:2149; 67. J Cell Biol (1996) 135:1741; 68. Biochim Biophys Acta (1993) 1147:194; 69. Fluorescent and Luminescent Probes for Biological Activity, Mason WT, Ed. (1993) p. 420; 70. Am J Physiol (1991) 260:C1; 71. FASEB J (1991) 5:2078; 72. Biophys J (1990) 57:1199; 73. Cytometry (2000) 39:151; 74. Biochim Biophys Acta (1991) 1067:71; 75. Plant Physiol (1990) 94:729; 76. J Photochem Photobiol A (1999) 121:177; 77. Anal Biochem (1991) 198:228; 78. Heteroatomic Chem (1990) 1:389; 79. Optics Comm (1989) 70:425; 80. Cytometry (1994) 17:151; 81. Biophys J (1996) 71:2656; 82. Electrophoresis (2003) 24:3253; 83. Methods Mol Biol (1999) 122:185; 84. Neuron (1998) 20:1081; 85. J Agric Food Chem (2000) 48:1150; 86. Methods Cell Biol (1999) 59:179; 87. Dev Dyn (2007) 232:359; 88. Biochim Biophys Acta (1993) 1149:86; 89. Eur J Cell Biol (1994) 65:172; 90. J Histochem Cytochem (1997) 45:743; 91. J Cell Biol (1993) 123:1567; 92. Exp Cell Res (1992) 199:29; 93. J Cell Biol (1989) 108:2201; 94. J Chromatogr (1987) 421:136; 95. Histochemistry (1992) 97:349; 96. Anal Chem (1990) 62:615; 97. Anal Biochem (1987) 167:228; 98. J Biol Chem (1995) 270:6357; 99. Anal Biochem (1993) 208:121; 100. Biotechniques (1996) 21:625; 101. J Biol Chem (2009) 284:3049; 102. J Virol (2008) 82:7624; 103. J Biol Chem (2009) 284:2383; 104. FEBS Lett (2007) 581:1688; 105. Photochem Photobiol (1993) 58:499; 106. Photochem Photobiol (1999) 70:557; 107. Biochemistry (1988) 27:399; 108. Biochemistry (1989) 28:8358; 109. Biochemistry (1990) 29:11134; 110. Biophys J (1994) 66:763; 111. Biophys J (1991) 60:179; 112. Anal Chem (2001) 73:2302; 113. Biophys J (2000) 78:290; 114. Biophys J (1999) 77:2090; 115. Biophys J (1997) 72:2413; 116. Biophys J (2001) 80:1417; 117. Biophys J (1994) 66:120; 118. Photochem Photobiol (1993) 57:420; 119. Biophys J (1990) 57:1179; 120. J Biol Chem (1990) 265:20044; 121. Photochem Photobiol (1993) 57:403; 122. Biochemistry (1992) 31:1550; 123. Biophys J (1990) 57:925; 124. Biochim Biophys Acta (2001) 1511:330; 125. Biophys J (1995) 68:1895; 126. Biophys J (1993) 65:1404; 127. Biochemistry (1992) 31:9473; 128. J Biol Chem (1994) 269:10298; 129. J Biol Chem (1994) 269:7429; 130. Biochemistry (1998) 37:7167; 131. Biochem J (1993) 290:411; 132. Eur J Biochem (1992) 204:127; 133. Nature (1986) 319:70; 134. J Photochem Photobiol A (2000) 131:95; 135. J Biomol Screen (2001) 6:429; 136. Biochim Biophys Acta (1982) 694:1; 137. Biophys J (1998) 74:422; 138. Biochemistry (1968) 7:3381; 139. Biochemistry (1999) 38:2110; 140. Biochemistry (1998) 37:4621; 141. Biochemistry (1998) 37:13862; 142. Biochemistry (1998) 37:16802; 143. J Chromatogr A (1994) 680:233; 144. Arch Biochem Biophys (1989) 268:239; 145. Biochemistry (1985) 24:3852; 146. Biochemistry (1985) 24:2034; 147. Biochemistry (1969) 8:3915; 148. Biochemistry (1992) 31:2982; 149. Biochim Biophys Acta (1990) 1040:66; 150. Proc Natl Acad Sci U S A (1977) 74:2334; 151. Biochemistry (1998) 37:4687; 152. Biochemistry (1994) 33:11891; 153. Biochemistry (1998) 37:17571; 154. Biochemistry (1992) 31:6470; 155. J Biol Chem (1984) 259:14647; 156. Biochemistry (1995) 34:7443; 157. Anal Biochem (1992) 204:110; 158. Biochemistry (1989) 28:6678; 159. Biochemistry (1993) 32:7589; 160. Chem Phys (1993) 169:351; 161. Biochemistry (1986) 25:6114. ™ The Handbook: A Guide to Fluorescent Probes and Labeling Technologies TheMolecular MolecularProbes Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes 585 Chapter 13 — Probes for Lipids and Membranes Section 13.5 Other Nonpolar and Amphiphilic Probes DATA TABLE 13.5 OTHER NONPOLAR AND AMPHIPHILIC PROBES Cat. No. MW Storage Soluble Abs EC Em Solvent Notes A47 299.34 L pH >6, DMF 372 7800 480 MeOH 1 A50 299.34 L DMF 319 27,000 422 MeOH 1 B153 672.85 L pH >6 395 23,000 500 MeOH 1, 2 344 80,000 378 MeOH 3 B311 444.57 L MeCN, CHCl3 665 161,000 676 MeOH 4 B3932 448.32 F,L DMSO, CHCl3 B30600 233.23 F,D,L DMSO 375 6000 458 MeOH C34556 438.25 F,D,L DMSO 588 68,000 616 MeOH 5 D109 529.63 L DMSO, EtOH 495 85,000 518 MeOH 6 D202 232.32 L DMF, MeCN 350 88,000 452 MeOH 7, 8 D250 353.55 L DMF, MeCN 364 20,000 497 MeOH 9 D3921 248.08 F,L EtOH, DMSO 502 98,000 510 MeOH 4 D3922 262.11 F,L EtOH, DMSO 493 89,000 504 MeOH 4 D3923 249.31 L DMF, DMSO 456 61,000 493 MeOH 358 25,000 517 MeOH 10 D12800 366.37 L DMSO, H2O F3857 584.79 L DMSO, EtOH 504 95,000 525 MeOH 6 H110 585.74 L DMSO, EtOH 497 92,000 519 MeOH 6 H22730 400.60 L DMSO, EtOH 366 20,000 453 MeOH 6 H34475 ~300 F,L DMSO 495 94,000 505 MeOH 5, 13 H34476 ~400 F,L DMSO 574 62,000 609 MeOH 5, 13 H34477 ~350 F,L DMSO 626 68,000 648 MeOH 5, 13 N1142 318.37 L DMF, DMSO 552 45,000 636 MeOH 11 O246 731.50 F,DD,L DMSO, EtOH 556 125,000 578 MeOH 12 P248 227.31 L DMF, MeCN 363 19,000 497 MeOH 9 T53 335.35 L DMF 318 26,000 443 MeOH 1 T204 461.62 D,L DMF, DMSO 355 75,000 430 MeOH 7 For definitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages. Notes 1. Fluorescence quantum yields of ANS and its derivatives are environment dependent and are particularly sensitive to the presence of water. QY of A47 is about 0.4 in EtOH, 0.2 in MeOH and 0.004 in water. Em is also somewhat solvent dependent. (Biochim Biophys Acta (1982) 694:1) 2. B153 is soluble in water at 0.1–1.0 mM after heating. 3. Absorption spectra of bis-pyrenyl alkanes have additional peaks at ~325 nm and <300 nm. Emission spectra include both monomer (~380 nm and ~400 nm) and excimer (~470 nm) peaks. 4. The absorption and fluorescence spectra of BODIPY® derivatives are relatively insensitive to the solvent. 5. This product is supplied as a ready-made solution in the solvent indicated under "Soluble." 6. Spectra of this product are pH dependent. Data listed are for basic solutions prepared in methanol containing a trace of KOH. 7. Diphenylhexatriene (DPH) and its derivatives are essentially nonfluorescent in water. Absorption and emission spectra have multiple peaks. The wavelength, resolution and relative intensity of these peaks are environment dependent. Abs and Em values are for the most intense peak in the solvent specified. 8. Stock solutions of DPH (D202) are often prepared in in tetrahydrofuran (THF). Long-term storage of THF solutions is not recommended because of possible peroxide formation in that solvent. 9. The emission spectrum of P248 is solvent dependent. Em = 401 nm in cyclohexane, 440 nm in CHCl3, 462 nm in MeCN, 496 nm in EtOH and 531 nm in H2O. (Biochemistry (1979) 18:3075) Abs is only slightly solvent dependent. The emission spectra of D250 in these solvents are similar to those of P248. 10. Em = 520 nm when bound to phospholipid bilayer membranes. Fluorescence in H2O is weak (Em ~600 nm). 11. The absorption and fluorescence spectra and fluorescence quantum yield of N1142 are highly solvent dependent. (J Lipid Res (1985) 26:781, Anal Biochem (1987) 167:228) 12. This product is intrinsically a sticky gum at room temperature. 13. Abs/Em in trioctanylglycerol = 498/507 nm, 582/616 nm and 635/652 nm for H34475, H34476 and H34477 respectively. The MolecularProbes® Probes Handbook: Handbook: A Probesand andLabeling LabelingTechnologies Technologies The Molecular A Guide Guide to to Fluorescent Fluorescent Probes ™ 586 IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes Chapter 13 — Probes for Lipids and Membranes Section 13.5 Other Nonpolar and Amphiphilic Probes PRODUCT LIST 13.5 OTHER NONPOLAR AND AMPHIPHILIC PROBES Cat. No. Product A47 1,8-ANS (1-anilinonaphthalene-8-sulfonic acid) *high purity* Quantity 100 mg A50 2,6-ANS (2-anilinonaphthalene-6-sulfonic acid) 100 mg B30600 Bimane azide B153 bis-ANS (4,4’-dianilino-1,1’-binaphthyl-5,5’-disulfonic acid, dipotassium salt) 5 mg B3932 (E,E)-3,5-bis-(4-phenyl-1,3-butadienyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY® 665/676) B311 1,3-bis-(1-pyrenyl)propane 10 mg 5 mg 25 mg C34556 CellTrace™ BODIPY® TR methyl ester *lipophilic counterstain for GFP* *solution in DMSO* D12800 Dapoxyl® sulfonic acid, sodium salt 1 mL 10 mg D3923 DCVJ (4-(dicyanovinyl)julolidine) 25 mg D3922 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY® 493/503) 10 mg D3921 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY® 505/515) D109 5-dodecanoylaminofluorescein 100 mg 10 mg D250 6-dodecanoyl-2-dimethylaminonaphthalene (laurdan) 100 mg D202 DPH (1,6-diphenyl-1,3,5-hexatriene) 100 mg F3857 Fluorescein octadecyl ester 10 mg H34477 HCS LipidTOX™ Deep Red neutral lipid stain *solution in DMSO* *for cellular imaging* 125 µL H34475 HCS LipidTOX™ Green neutral lipid stain *solution in DMSO* *for cellular imaging* 125 µL H34476 HCS LipidTOX™ Red neutral lipid stain *solution in DMSO* *for cellular imaging* 125 µL H22730 4-heptadecyl-7-hydroxycoumarin 10 mg H110 5-hexadecanoylaminofluorescein 100 mg N1142 Nile red 25 mg O246 Octadecyl rhodamine B chloride (R18) 10 mg P248 Prodan (6-propionyl-2-dimethylaminonaphthalene) T204 TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate) T53 2,6-TNS (2-(p-toluidinyl)naphthalene-6-sulfonic acid, sodium salt) 100 mg 25 mg 100 mg ™ The Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies TheMolecular Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPORTANT NOTICE:described The products described thiscovered manual are by oneLimited or moreUse Limited Use Label License(s). to the Appendix IMPORTANT NOTICE : The products in this manualinare by covered one or more Label License(s). PleasePlease referrefer to the Appendix on on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes thermofisher.com/probes 587