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SIXTEEN
CHAPTER 16
Probes for Endocytosis, Receptors
and Ion Channels
16.1 Probes for Following Receptor Binding and Phagocytosis.
. . . . . . . . . . . . . . . . . . . . . . . . . . 741
Ligands for Studying Receptor-Mediated Endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 741
Fc OxyBURST® Green Assay Reagent: Fluorogenic Immune Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 741
OxyBURST® Green H2HFF BSA Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 742
Amine-Reactive OxyBURST® Green Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 742
Fluorescent Low-Density Lipoprotein Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 742
Fluorescent Acetylated LDL Complexes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 743
Fluorescent Lipopolysaccharides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 744
Epidermal Growth Factor Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 744
Transferrin Conjugates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 745
Fluorescent Fibrinogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 746
DQ™ Ovalbumin: A Probe for Antigen Processing and Presentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747
Fluorescent Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747
Fluorescent Casein. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747
Fluorescent Chemotactic Peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747
Fluorescent Insulin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748
Fluorescent Dexamethasone Probe for Glucocorticoid Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748
Fluorescent Histone H1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748
Fluorescent Probes for the Acrosome Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748
Methods for Detecting Internalized Fluorescent Ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748
Membrane Markers of Endocytosis and Exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 749
FM® 1-43. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 749
Other Analogs of FM® 1-43 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750
FM® 1-43FX and FM® 4-64FX: Fixable FM® Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750
4-Di-1-ASP and 4-Di-2-ASP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750
TMA-DPH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750
Fluorescent Cholera Toxin Subunit B: Markers of Lipid Rafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750
Fluorescent Protein–Based Lipid Raft Markers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 751
Fluorescent Protein–Based Synaptic Vesicle Markers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 751
Anti–Synapsin I Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 752
High Molecular Weight Polar Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 752
Fluorescent Protein–Based Endosomal Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 752
BioParticles® Fluorescent Bacteria and Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 753
Vybrant® Phagocytosis Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 753
pHrodo™ BioParticles® Fluorescent Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 753
pHrodo™ Phagocytosis Particle Labeling Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 754
Opsonizing Reagents and Nonfluorescent BioParticles® Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 754
Fluorescent Polystyrene Microspheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 754
Fluorescent Microspheres Coated with Collagen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 754
Fluorescent Dextrans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 755
pH Indicator Dextrans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 755
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739
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
Low Molecular Weight Polar Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 755
Data Table 16.1 Probes for Following Receptor Binding and Phagocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 757
Product List 16.1 Probes for Following Receptor Binding and Phagocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 758
16.2 Probes for Neurotransmitter Receptors
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760
α-Bungarotoxin Probes for Nicotinic Acetylcholine Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760
Fluorescent α-Bungarotoxins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760
Biotinylated α-Bungarotoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760
Unlabeled α-Bungarotoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 761
Amplex® Red Acetylcholine/Acetylcholinesterase Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 761
BODIPY® FL Prazosin for α1-Adrenergic Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 762
BODIPY® TMR-X Muscimol for GABAA Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 762
Fluorescent Angiotensin II for AT1 and AT2 Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 762
Naloxone Fluorescein for µ-Opioid Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763
Probes for Amino Acid Neurotransmitter Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763
Caged Amino Acid Neurotransmitters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763
Anti–NMDA Receptor Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763
Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763
Probes for Other Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 764
Data Table 16.2 Probes for Neurotransmitter Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 765
Product List 16.2 Probes for Neurotransmitter Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 765
16.3 Probes for Ion Channels and Carriers
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 766
2+
Probes for Ca Channels and Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 766
Fluorescent Dihydropyridine for L-Type Ca2+ Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 766
BODIPY® FL Verapamil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 766
Eosin Derivatives: Inhibitors of the Calcium Pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 767
Premo™ Cameleon Calcium Sensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 767
Probes for Na+ Channels and Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768
Amiloride Analogs: Probes for the Na+ Channel and the Na+/H+ Antiporter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768
Ouabain Probes for Na+/K+-ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768
Using BacMam Technology to Deliver and Express Sodium Channel cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768
Probes for K+ Channels and Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768
Glibenclamide Probes for the ATP-Dependent K+ Channel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768
FluxOR™ Potassium Ion Channel Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 769
Using BacMam Technology to Deliver and Express Potassium Channel cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 770
Probes for Anion Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 770
Stilbene Disulfonates: Anion-Transport Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 770
DiBAC4(5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 771
Eosin Maleimide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 771
Premo™ Halide Sensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 771
Data Table 16.3 Probes for Ion Channels and Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 772
Product List 16.3 Probes for Ion Channels and Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 772
The
Guideto
toFluorescent
Fluorescent Probes
Probes and
TheMolecular
MolecularProbes®
Probes Handbook:
Handbook: AAGuide
and Labeling
LabelingTechnologies
Technologies
™
740
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 products
on page 975.
Productsin
arethis
Formanual
Researchare
Usecovered
Only. Notbyintended
for anyLimited
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or diagnostic
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License(s).
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Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
Section 16.1 Probes for Following Receptor Binding and Phagocytosis
16.1 Probes for Following Receptor Binding and Phagocytosis
The plasma membrane defines the inside and outside of the cell. It not only encloses the
cytosol to maintain the intracellular environment but also serves as a formidable barrier to the
extracellular environment. Because cells require input from their surroundings—in the form
of hydrated ions, small polar molecules, large biomolecules and even other cells—they have
developed strategies for overcoming this barrier. Some of these mechanisms involve initial
formation of receptor–ligand complexes, followed by transport of the ligand across the cell
membrane.1–5
This section focuses on probes for following receptor binding, endocytosis and exocytosis.
Section 16.2 describes tools for studying neurotransmitter receptors, which mediate external
chemical messenger control over the electrical activity of neurons. Section 16.3 discusses strategies for monitoring ion channels and carriers, which are the molecular centerpiece of neural
transmission and bioenergetics.
Figure 16.1.1 2’,7’-dichlorodihydrofluorescein diacetate
(2’,7’-dichlorofluorescin diacetate; H2DCFDA, D399).
H2DCF-BSA
immune complex
Fc receptors
Ligands for Studying Receptor-Mediated Endocytosis
We offer a variety of fluorescent and fluorogenic ligands that bind to membrane receptors
and are subsequently internalized. In some cases, the bound ligand is released intracellularly and
the receptor is then recycled to the plasma membrane. Receptor binding may also result in signal
transduction (Chapter 17), Ca 2+ mobilization (Chapter 19), intracellular pH changes (Chapter 20)
and formation of reactive oxygen species (ROS, Chapter 18).
When soluble or surface-bound IgG immune complexes interact with Fc receptors on phagocytic cells, a number of host defense mechanisms are activated, including phagocytosis and activation of an NADPH oxidase–mediated oxidative burst.6 Dichlorodihydrofluorescein diacetate
(H2DCFDA, D399; Section 18.2; Figure 16.1.1), a cell-permeant fluorogenic probe that localizes in
the cytosol, has frequently been used to monitor this oxidative burst.7 Its fluorescence response,
however, is limited by the diffusion rate of the reactive oxygen species into the cytosol from the
phagovacuole where it is generated. In contrast, Fc OxyBURST® assay reagents permit direct
measurement of the kinetics of Fc receptor–mediated internalization and the subsequent oxidative burst in the phagovacuole, yielding signals that are many times brighter than those generated
by H2DCFDA (Figure 16.1.2, Figure 16.1.3).
Fc OxyBURST® Green assay reagent (F2902) was developed in collaboration with Elizabeth
Simons of Boston University to monitor the oxidative burst in phagocytic cells using fluorescence
instrumentation. Fc OxyBURST® Green assay reagent comprises bovine serum albumin (BSA)
that has been covalently linked to dichlorodihydrofluorescein (H2DCF) and then complexed
with a purified rabbit polyclonal anti-BSA antibody (A11133). When these immune complexes
bind to Fc receptors, the nonfluorescent H2DCF molecules are internalized within the phagovacuole and subsequently oxidized to green-fluorescent 2’,7’-dichlorofluorescein (DCF; Figure
16.1.2, Figure 16.1.3). Unlike H2DCFDA, Fc OxyBURST® Green assay reagent does not require
intracellular esterases for activation, making this reagent particularly suitable for detecting the
oxidative burst in cells with low esterase activity such as monocytes.8 Fc OxyBURST® Green assay
reagent reportedly produces >8 times more fluorescence than does H2DCFDA at 60 seconds and
>20 times more at 15 minutes following internalization of the immune complex.9
Published reports have described the use of Fc OxyBURST® Green assay reagent to study the
oxidative burst in phagovacuoles.10–12 Neutrophils from patients with chronic granulomatous
disease, a genetic deficiency known to disable NADPH oxidase–mediated oxidative bursts, were
Figure 16.1.2 Fc OxyBURST® Green assay reagent (F2902)
for fluorescent detection of the Fc receptor–mediated
phagocytosis pathway. Dichlorodihydrofluorescein (H2DCF)
is covalently attached to bovine serum albumin (BSA), then
complexed with a rabbit polyclonal anti-BSA antibody
(A11133). Upon binding to an Fc receptor, the nonfluorescent immune complex is internalized and subsequently oxidized to the fluorescent DCF.
Fluorescence
Fc OxyBURST® Green Assay Reagent: Fluorogenic Immune Complex
phagosome
H2DCF-BSA
immune complex
H2DCFDA
0
200
400
600
Time (seconds)
800
1000
Figure 16.1.3 Fluorescence emission of human neutrophils
challenged either with Fc OxyBURST® Green assay reagent
(H2DCF-BSA immune complexes, F2902) or with unlabeled
immune complexes in the presence of dichlorodihydrofluorescein diacetate (H2DCFDA; D399). Fc OxyBURST® Green assay reagent generates significantly more fluorescence than
does the more commonly used H2DCFDA. Flow cytometry
data provided by Elizabeth Simons, Boston University.
™
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the Appendix
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Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
observed to bind but not oxidize Fc OxyBURST® Green assay reagent 9 (Figure 16.1.4). Using
microfluorometry to detect the Fc OxyBURST® Green signal, researchers were able to simultaneously monitor oxidative activity and membrane currents in voltage-clamped human mononuclear cells.13
Control
Fluorescence
Section 16.1 Probes for Following Receptor Binding and Phagocytosis
OxyBURST® Green H2HFF BSA Reagent
CGD
0
50
100
150
200
Time (seconds)
Figure 16.1.4 Oxidative bursts of human neutrophils from a
healthy donor (control) compared with those from a patient
with chronic granulomatous disease (CGD), as detected using
the Fc OxyBURST® Green assay reagent (F2902). Flow cytometry data provided by Elizabeth Simons, Boston University.
DQ™ BSA
immune complex
Fc receptors
phagosome
Figure 16.1.5 Immune complex of DQ™ BSA conjugate
(D12050, D12051) with an anti–bovine serum albumin (BSA)
antibody (A11133) for the fluorescent detection of the Fc receptor–mediated phagocytosis pathway. The DQ™ BSA is a
derivative of BSA that is labeled to such a high degree with
either the green-fluorescent BODIPY® FL or red-fluorescent
BODIPY® TR-X dye that the fluorescence is self-quenched.
Upon binding to an Fc receptor, the nonfluorescent immune
complex is internalized and the protein is subsequently hydrolyzed to fluorescent peptides within the phagovacuole.
Figure 16.1.6 2’,7’-dichlorodihydrofluorescein diacetate, succinimidyl ester (OxyBURST® Green H2DCFDA, SE, D2935).
OxyBURST® Green H2HFF BSA reagent 14–16 (O13291) is similar to Fc OxyBURST® Green
assay reagent, except that it is prepared by reacting the succinimidyl ester of a reduced form of
our Oregon Green® 488 dye with BSA. The absorption maximum of the oxidation product of this
reagent (~492 nm) matches the 488 nm spectral line of the argon-ion laser better than does that
of Fc OxyBURST® Green assay reagent (~495 nm). OxyBURST® Green H2HFF BSA reagent can
also be complexed with anti-BSA antibody to form an immune complex that can be utilized like
the Fc OxyBURST® Green assay reagent (F2902).
All of the OxyBURST® reagents are slowly oxidized by molecular oxygen and are also susceptible to oxidation catalyzed by illumination in a fluorescence microscope. These reagents are reasonably stable in solution for at least six months when stored under nitrogen or argon in the dark
at 4°C. We also offer a purified rabbit polyclonal anti-BSA antibody (A11133), which can bind
any of our BSA conjugates (Section 14.7) or fluorogenic DQ™ BSA conjugates (D12050, D12051;
Section 10.4) to create immune complexes for analyzing the Fc receptor–mediated phagocytosis pathway. In the case of the anti-BSA antibody complex with DQ™ BSA, initial binding and
internalization of the probe is followed by hydrolysis to fluorescent peptides by proteases in the
phagovacuole 17 (Figure 16.1.5).
Amine-Reactive OxyBURST® Green Reagent
As an alternative to Fc OxyBURST® Green assay reagent and OxyBURST® Green H2HFF
BSA, we offer amine-reactive OxyBURST® Green H2DCFDA succinimidyl ester (2’,7’-dichlorodihydrofluorescein diacetate, SE; D2935; Figure 16.1.6), which can be used to prepare oxidation-sensitive conjugates of a wide variety of biomolecules and particles, including antibodies, antigens, peptides, proteins, dextrans, bacteria, yeast and polystyrene microspheres.9,18,19
Following conjugation to amines, the two acetates of OxyBURST® Green H2DCFDA reagent can
be removed by treatment with hydroxylamine at near-neutral pH to yield the oxidant-sensitive
dichlorodihydrofluorescein conjugates. Thus, like our Fc OxyBURST® Green assay reagent, they
provide a means of detecting the oxidative burst in phagocytic cells.18
Several other reagents—dihydrofluoresceins, dihydrorhodamines, dihydroethidium and
chemiluminescent probes—that have been used to detect the reactive oxygen species (ROS) produced during phagocytosis are described in Section 18.2.
Fluorescent Low-Density Lipoprotein Complexes
The human LDL complex, which delivers cholesterol to cells by receptor-mediated endocytosis, comprises a core of about 1500 molecules of cholesteryl ester and triglyceride, surrounded
by a 20 Å–thick shell of phospholipids, unesterified cholesterol and a single copy of apoprotein B
100 20 (MW ~500,000 daltons). Once internalized, LDL dissociates from its receptor and eventually appears in lysosomes.21 In addition to unlabeled LDL (L3486), which has been reported to
be an effective vehicle for selectively delivering antitumor drugs to cancer cells, 22 we offer two
classes of labeled LDL probes—those containing an unmodified apoprotein, used to study the
mechanisms of normal cholesterol delivery and internalization, and those with an acetylated
apoprotein, used to study endothelial, microglial and other cell types that express receptors that
specifically bind this modified LDL.
For the class of labeled LDL probes containing unmodified apoprotein, we prepare LDL noncovalently labeled with either DiI (DiI LDL, L3482) or the BODIPY® FL fluorophore (BODIPY®
FL LDL, L3483), highly fluorescent lipophilic dyes that diffuse into the hydrophobic portion of
the LDL complex without affecting the LDL-specific binding of the apoprotein. As compared
with DiI LDL, BODIPY® FL LDL is more efficiently excited by the 488 nm spectral line of the
argon-ion laser, making it better suited for flow cytometry and confocal laser-scanning microscopy studies. Like our BODIPY® FL C5-ceramide (D3521, Section 12.4), BODIPY® FL LDL may
exhibit concentration-dependent long-wavelength emission (>550 nm), precluding its use for
The
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andLabeling
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The
Molecular
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to Fluorescent
Fluorescent Probes
™
742
IMPORTANT
NOTICE:
The products
described
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coveredare
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Limited
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Please
refer to thePlease
Appendix
onto
IMPORTANT
NOTICE
: The products
described
in thisaremanual
by one
or more
Limited
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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
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Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
Section 16.1 Probes for Following Receptor Binding and Phagocytosis
multicolor labeling with red fluorophores. Both DiI LDL and BODIPY® FL LDL have been used
to investigate the binding specificity and partitioning of LDL throughout the Schistosoma mansoni parasite 23 (Figure 16.1.7). Fluorescent LDL complexes have also proven useful in a variety
of experimental systems to:
• Count the number of cell-surface LDL receptors, analyze their motion and clustering and
follow their internalization 24–26
• Demonstrate that fibroblasts grown continuously in the presence of DiI LDL (L3482) proliferate normally and exhibit normal morphology, 27 making DiI LDL a valuable alternative
to 125I-labeled LDL for quantitating LDL receptor activity 28
• Identify LDL receptor–deficient Chinese hamster ovary (CHO) cell mutants 29
• Image LDL receptor endocytosis in COS7 cells expressing Green Fluorescent Protein (GFP)–
tagged GTPase 30
• Investigate the modulation of LDL receptor expression by statin drugs 31,32
Figure 16.1.7 DiI LDL (L3482) bound to the surface and
internalized in the gut of the parasite Schistosoma mansoni.
The distribution of LDL in the parasite is used to study a putative mechanism by which the parasite may avoid host immune recognition. Image contributed by John P. Caulfield,
Harvard School of Public Health.
We prepare fluorescent LDL products from fresh human plasma, and they should be stored
refrigerated and protected from light; LDL products must not be frozen. Because preparation of
these complexes involves several variables, some batch-to-batch variability in degree of labeling
and fluorescence yield is expected.
Fluorescent Acetylated LDL Complexes
If the lysine residues of LDL’s apoprotein have been acetylated, the LDL complex no longer binds to the LDL receptor, 33 but rather is taken up by macrophage and endothelial cells
that possess “scavenger” receptors specific for the modified LDL. 34,35 Once the acetylated LDL
(AcLDL) complexes accumulate within these cells, they assume an appearance similar to that of
foam cells found in atherosclerotic plaques. 36–38 We offer unlabeled AcLDL (L35354), as well as
AcLDL noncovalently labeled with DiI L(3484) and AcLDL covalently labeled with Alexa Fluor®
488 dye (L23380), Alexa Fluor® 594 dye (L35353) or BODIPY® FL dye (L3485). Fluorescent dye
conjugates of high-density lipoproteins, including one prepared using Alexa Fluor® 488 succinimidyl ester (A20000, A20100; Section 1.3), are taken up via the same receptor as acetylated
LDL complexes. 39
Using DiI AcLDL, researchers have discovered that the scavenger receptors on rabbit fibroblasts and smooth muscle cells appear to be up-regulated through activation of the protein
kinase C pathway.40 DiI AcLDL has also been used to show that Chinese hamster ovary (CHO)
cells express AcLDL receptors that are distinct from macrophage scavenger receptors.41,42
Ultrastructural localization of endocytic compartments that maintain a connection to the extracellular space has been achieved by photoconversion of DiI AcLDL in the presence of diaminobenzidine 43 (Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents—Note
14.2). A quantitative assay for LDL- and scavenger-receptor activity in adherent and nonadherent cultured cells that avoids the use of both radioactivity and organic solvents has been
described.44
It has now become routine to identify endothelial cells and microglial cells in primary cell
culture by their ability to take up DiI AcLDL 45,46 (Figure 16.1.8). DiI AcLDL was employed in
order to confirm endothelial cell identity in investigations of shear stress 47 and P-glycoprotein
expression,48 as well as to identify blood vessels in a growing murine melanoma.49 In addition,
patch-clamp techniques have been used to investigate membrane currents in mouse microglia,
which were identified both in culture and in brain slices by their staining with DiI AcLDL. 50,51
For some applications, Alexa Fluor® 488, Alexa Fluor® 594 and BODIPY® FL AcLDL may be the
preferred probes because the dyes are covalently bound to the modified apoprotein portion of
the LDL complex and are therefore not extracted during subsequent manipulations of the cells.
Furthermore, the green-fluorescent Alexa Fluor® 488 AcLDL has spectral characteristics similar
to fluorescein and is useful for analyses with instruments equipped with the 488 nm argon-ion
laser excitation sources, including flow cytometers and confocal laser-scanning microscopes. The
bright and photostable red-fluorescent Alexa Fluor® 594 AcLDL conjugate is useful for multilabeling experiments with green-fluorescent probes and can be efficiently excited by the 594 nm
spectral line of the orange He-Ne laser.52
Figure 16.1.8 Microglial cells in a rat hippocampus cryosection labeled with red-orange–fluorescent DiI acetylated
low-density lipoprotein (L3484) and stained using blue-fluorescent DAPI (D1306, D3571, D21490).
™
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 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.
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743
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
HO
Fluorescent Lipopolysaccharides
OH
O
O
P
O
O
OH
O
O
O
O
NH HO
O
NH O
O
P
OH
•
•
•
•
•
O
O
O
HO
O
O
160
120
Epidermal Growth Factor Derivatives
200
Counts
160
120
80
40
0
100
101
102
103
104
Green fluorescence
Counts
B
Alexa Fluor® 488 LPS from E. coli serotype 055:B5 (A23351)
Alexa Fluor® 488 LPS from S. minnesota (A23356)
Alexa Fluor® 568 LPS from E. coli serotype 055:B5 (A23352)
Alexa Fluor® 594 LPS from E. coli serotype 055:B5 (A23353)
BODIPY® FL LPS from E. coli serotype 055:B5 (A23350)
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 is the key initiation step in the mammalian immune response to infection by gram-negative
bacteria.53 The structural core of LPS, and the primary determinant of its biological activity, is an
N-acetylglucosamine derivative, lipid A (Figure 16.1.9). 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.
The fluorescent BODIPY® FL and Alexa Fluor® LPS conjugates, which are labeled with succinimidyl esters of these dyes, allow researchers to follow LPS-elicited inflammatory responses.53,54 Lipopolysaccharide internalization activates endotoxin-dependent signal transduction
in cardiomyocytes.55 Alexa Fluor® 488 LPS conjugates (L23351, L23356) have been shown to
selectively label microglia in a mixed culture containing oligodendrocyte precursors, astrocytes
and microglia.56
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 lipoprotein 57 (HDL). Another study
utilized a BODIPY® FL derivative of LPS from S. minnesota to demonstrate transport to the Golgi
apparatus in neutrophils, 58,59 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.60,61 Cationic lipids are reported to assist the translocation of fluorescent lipopolysaccharides
into live cells; 62 cell surface–bound LPS can be quenched by trypan blue.57
Other probes useful for analyzing lipopolysaccharides include fluorescent analogs of the
LPS-binding antibiotic polymyxin B (Section 17.3) and BODIPY® TR cadaverine (D6251, Section
3.4). BODIPY® TR cadaverine binds with high selectivity to lipid A, forming the basis for highthroughput ligand displacement assays for identifying endotoxin antagonists.63,64
Figure 16.1.9 Structure of the lipid A component of
Salmonella minnesota lipopolysaccharide.
A
We offer fluorescent conjugates of lipopolysaccharides (LPS) from Escherichia coli and
Salmonella minnesota (Table 16.1), including:
OH
O
O
O
Section 16.1 Probes for Following Receptor Binding and Phagocytosis
200
80
40
0
100
101
102
103
104
Green fluorescence
Figure 16.1.10 Detection of epidermal growth factor (EGF)
receptors directly or with signal amplification. Cells expressing high (A431 cells, panel A) and low (NIH 3T3 cells, panel
B) levels of EGF receptors were either directly labeled with
the preformed Alexa Fluor® 488 complex of biotinylated
epidermal growth factor (E13345, blue) or indirectly labeled
with biotinylated EGF (E3477) followed by either Alexa
Fluor® 488 streptavidin (S11223, green) or HRP-conjugated
streptavidin and Alexa Fluor® 488 tyramide (purple), components of our TSA™ Kit #22 (T20932).
Epidermal growth factor (EGF) is a 53–amino acid polypeptide hormone (MW 6045 daltons)
that stimulates division of epidermal and other cells. The EGF receptors include the HER-2/
neu receptor (where “HER-2” is an acronym for human epidermal growth factor receptor-2 and
“neu” refers to an original mouse origin); HER-2/neu overexpression has evolved as a prognostic/
predictive factor in some solid tumors.65–67 Binding of EGF to its 170,000-dalton receptor protein results in the activation of kinases, phospholipases and Ca 2+ mobilization and precipitates
a wide variety of cellular responses related to differentiation, mitogenesis, organ development
and cell motility.
We offer unlabeled mouse submaxillary gland EGF (E3476), as well as the following EGF
conjugates, each containing a single fluorophore or biotin on the N-terminal amino acid:
•
•
•
•
Fluorescein EGF (E3478)
Oregon Green® 514 EGF (E7498)
Tetramethylrhodamine EGF (E3481)
Biotin-XX EGF (E3477)
The dissociation constant of the EGF conjugates in DMEM-HEPES medium is in the low
nanomolar range for human epidermoid carcinoma (A431) cells,68 a value that approximates
that of the unlabeled EGF. Fluorescently labeled EGF has enabled scientists to use fluorescence
resonance energy transfer techniques to assess EGF receptor–receptor and receptor–membrane
interactions 69–71 (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2). Using fluorescein
The
MolecularProbes®
Probes Handbook:
Handbook: AAGuide
and Labeling
LabelingTechnologies
Technologies
The
Molecular
Guideto
toFluorescent
Fluorescent Probes
Probes and
™
744
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
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Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
Section 16.1 Probes for Following Receptor Binding and Phagocytosis
Table 16.1 Fluorescent lipopolysaccharide conjugates.
Fluorophore
Abs *
Em *
Escherichia coli
Salmonella minnesota
Alexa Fluor® 488
495
519
L23351
L23356
BODIPY® FL
503
513 †
L23350
Alexa Fluor® 568
578
603
L23352
Alexa Fluor® 594
590
617
L23353
* Approximate absorption (Abs) and fluorescence emission (Em) maxima for conjugates, in nm. † At high concentrations, the
emission maximum for the BODIPY® FL dye may shift from ~513 nm to ~620 nm.1,2
1. J Immunol (1997) 158:3925; 2. J Biol Chem (1996) 271:4100.
EGF as the donor and tetramethylrhodamine EGF as the acceptor, researchers examined temperature-dependent lateral and transverse distribution of EGF receptors in A431 cell plasma
membranes.71 When fluorescein EGF binds to A431 cells, it apparently undergoes a biphasic
quenching, which can be attributed first to changes in rotational mobility upon binding and
then to receptor–ligand internalization. By monitoring this quenching in real time, the rate
constants for the interaction of fluorescein EGF with its receptor were determined.72 Although
fluorescently labeled EGF can be used to follow lateral mobility and endocytosis of the EGF
receptor,73,74 the visualization of fluorescent EGF may require low-light imaging technology or
Qdot® nanocrystals, especially in cells that express low levels of the EGF receptor.75 In cells with
few EGF receptors, it can be difficult to detect signal over background fluorescence unless signal
amplification methods are employed (Figure 16.1.10).
Biotin-XX EGF contains a long spacer arm that enhances the probe’s affinity for the EGF
receptor and facilitates binding of dye-, Qdot® nanocrystal– or enzyme-conjugated streptavidins 75–79 (Section 7.6). Using biotinylated EGF and phycoerythrin-labeled secondary reagents
(Section 6.4), researchers were able to detect as few as 10,000 EGF cell-surface receptors by confocal laser-scanning microscopy.80 Tyramide signal amplification (TSA) technology (Section 6.2)
is particularly valuable for detection and localization of low-abundance EGF receptors by both
imaging and flow cytometry (Figure 16.1.10). For additional sensitivity, we prepare biotinylated
EGF precomplexed to fluorescent streptavidin:
•
•
•
•
Biotinylated EGF complexed to Alexa Fluor® 488 streptavidin (E13345, Figure 16.1.11)
Biotinylated EGF complexed to Alexa Fluor® 555 streptavidin (E35350)
Biotinylated EGF complexed to Alexa Fluor® 647 streptavidin (E35351)
Biotinylated EGF complexed to Texas Red® streptavidin 81,82 (E3480)
These products yield several-fold brighter signals per EGF receptor when compared with the
direct conjugates. We have found that EGF receptors can easily be detected with these complexes
without resorting to low-light imaging technology (Figure 16.1.12). A quantitative high-content
screening (HCS) assay for EGF receptor modulators based on imaging the internalization of the
Alexa Fluor® 555 EGF complex internalization has been reported.83
Figure 16.1.12 Lightly fixed human epidermoid carcinoma cells (A431) stained with biotinylated epidermal growth
factor (EGF) complexed to Texas Red® streptavidin (E3480).
An identical cell preparation stained in the presence of a
100-fold excess of unlabeled EGF (E3476) showed no fluorescent signal.
Table 16.2 Transferrin conjugates.
Transferrin Conjugates
Transferrin is a monomeric serum glycoprotein (MW ~80,000 daltons) that binds up to two
Fe3+ atoms for delivery to vertebrate cells through receptor-mediated endocytosis. Once ironcarrying transferrin proteins are inside endosomes, the acidic environment favors dissociation
of the sequestered iron from the transferrin–receptor complex. Following the release of iron,
the apotransferrin is recycled to the plasma membrane, where it is released from its receptor to
scavenge more iron. Transferrin uptake is a prototypical and ubiquitous example of clathrinmediated endocytosis. Although transferrin uptake is widely regarded as a surrogate measure of
total clathrin-mediated endocytosis, perturbations that are specific to transferrin endocytosis
impel caution in making such extrapolations.2
Our fluorescent and biotinylated di-ferric (Fe3+) human transferrin conjugates (Table 16.2)
include:
•
•
•
•
Figure 16.1.11 Early endosomes in live HeLa cells identified after a 10-minute incubation with green-fluorescent
Alexa Fluor® 488 epidermal growth factor (E13345). The
cells were subsequently fixed with formaldehyde and labeled with an antibody to the late endosomal protein, RhoB.
That antibody was visualized with a red-orange–fluorescent
secondary antibody. Nuclei were stained with TO-PRO®-3
iodide (T3605, pseudocolored blue). The image was contributed by Harry Mellor, University of Bristol.
Fluorescein transferrin (T2871)
Alexa Fluor® 488 transferrin 84–87 (T13342)
Alexa Fluor® 546 transferrin 88,89 (T23364)
Alexa Fluor® 555 transferrin 90 (T35352)
Cat. No.
Label
Abs *
Em *
T2871
Fluorescein
494
518
T13342
Alexa Fluor® 488
495
518
T2872
Tetramethylrhodamine
555
580
T23364
Alexa Fluor® 546
556
575
T35352
Alexa Fluor® 555
555
565
T23365
Alexa Fluor® 568
578
603
T13343
Alexa Fluor® 594
589
616
T2875
Texas Red®
595
615
T23362
Alexa Fluor® 633
632
647
T23366
Alexa Fluor® 647
650
665
T35357
Alexa Fluor® 680
679
702
T23363
Biotin-XX
NA
NA
* Approximate absorption (Abs) and fluorescence
emission (Em) maxima for conjugates, in nm. NA = Not
applicable.
™
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
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Label
PleasePlease
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745
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
•
•
•
•
•
•
•
•
Section 16.1 Probes for Following Receptor Binding and Phagocytosis
Alexa Fluor® 568 transferrin 91 (T23365)
Alexa Fluor® 594 transferrin 92–94 (T13343, Figure 16.1.13)
Alexa Fluor® 633 transferrin (T23362)
Alexa Fluor® 647 transferrin 73,95,96 (T23366)
Alexa Fluor® 680 transferrin (T35357)
Tetramethylrhodamine transferrin (T2872)
Texas Red® transferrin (T2875)
Biotin-XX transferrin (T23363)
Alexa Fluor® transferrin conjugates are highly recommended because of their brightness,
enhanced photostability and lack of sensitivity to pH (Section 1.3). The pH sensitivity of fluorescein-labeled transferrin has been exploited to investigate events occurring during endosomal
acidification.97–100 Fluorescent transferrins have also been used to:
Figure 16.1.13 Live HeLa cells incubated with Alexa Fluor®
594 transferrin (T13343) for 10 minutes to label early endosomes. The cells were subsequently fixed with formaldehyde
and labeled with an antibody to the endosomal protein RhoD.
That antibody was visualized with a green-fluorescent secondary antibody. Yellow fluorescence indicates regions of co-localization. To illustrate the staining pattern, the cells were imaged
by both fluorescence (top panel) and differential interference
contrast (DIC) microscopy (bottom panel). The image was contributed by Harry Mellor, University of Bristol.
Alexa Fluor® 488 fibrinogen
104
Unactivated
3
10
102
101
100
101
102
103
104
102
103
104
Alexa Fluor® 488 fibrinogen
104
Activated
3
10
102
101
100
100
Uptake of a horseradish peroxidase (HRP) conjugate of transferrin by endosomes has been
detected using tyramide signal amplification (TSA, Section 6.2) by catalytic deposition of biotin
tyramide and use of fluorescent streptavidin conjugates 105 (Section 7.6).
In addition to fluorescent and biotinylated transferrin conjugates, we offer a mouse monoclonal IgG1 anti–human transferrin receptor antibody (A11130). This antibody can be used with any
of our Zenon® Mouse IgG1 Labeling Kits (Section 7.3, Table 7.7) for rapid preparation of labeling
complexes. Antibodies against transferrin receptors have been used for indirect immunofluorescent staining of the transferrin receptor,106–108 transport of molecules across the blood–brain
barrier,109 characterization of transferrin in recycling compartments,106 enzyme-linked immunosorbent assays (ELISAs) 108 and antibody competition with transferrin uptake.110
Fluorescent Fibrinogen
0
10
• Analyze the role of the γ-chain of type III IgG receptors in antigen–antibody complex
internalization 101
• Characterize endocytic apparatus phenotypes in drug-resistant cancer cells 85
• Demonstrate that the fungal metabolite brefeldin A (B7450, Section 12.4) induces an increase
in tubulation of transferrin receptors in BHK-21 cells 102 and in the perikaryal–dendritic
region of cultured hippocampal neurons 103
• Image transferrin receptor dynamics using FRET 104
• Observe receptor trafficking in live cells by confocal laser-scanning microscopy 74
101
R-PE anti-CD41
Figure 16.1.14 Interaction of fluorescently labeled fibrinogen with activated platelets. Whole blood was first incubated with an R-phycoerythrin (R-PE)–labeled anti-CD41 antibody to label the platelets. 20 µM adenosine 5’-diphosphate
(ADP) was added in order to activate the platelets, then
2 µg/mL Alexa Fluor® 488 fibrinogen (F13191) was added
and incubated with the sample for 5 minutes. Cells were analyzed by flow cytometry using excitation at 488 nm. Both
activated and unactivated platelets show binding of the
anti-CD41 antibody; however, only the activated platelets
show strong binding by fibrinogen. A total of 5000 platelets
are shown in each experiment.
Fibrinogen is a key component in the blood clotting process and can support both platelet–
platelet and platelet–surface interactions by binding to the glycoprotein IIb-IIIa (GPIIb-IIIa)
receptor (also called integrin α IIbβ3) of activated platelets. Activation of GPIIb-IIIa is required
for fibrinogen binding, which leads to platelet activation, adhesion, spreading and microfilament
reorganization of human endothelial cells in vitro. Bone marrow transplant patients have significantly higher levels of fibrinogen binding, as compared with controls Soluble fibrinogen binds
to its receptor with a Ca 2+-dependent apparent Kd of 0.18 µM.111 This binding is mediated by the
tripeptide sequence Arg-Gly-Asp (RGD), found in both fibrinogen and fibronectin.
Fluorescently labeled fibrinogen has proven to be a valuable tool for investigating platelet activation and subsequent fibrinogen binding.112–114 Alexa Fluor® 647 fibrinogen has been used to identify
activated platelets by flow cytometry.115 The binding of fluorescein fibrinogen to activated platelets
has been shown to be saturable and can be inhibited completely by underivatized fibrinogen.116,117
We offer four conjugates of human fibrinogen in three different fluorescent colors:
•
•
•
•
Alexa Fluor® 488 human fibrinogen conjugate (F13191)
Oregon Green® 488 human fibrinogen conjugate (F7496)
Alexa Fluor® 546 human fibrinogen conjugate (F13192)
Alexa Fluor® 647 human fibrinogen conjugate (F35200)
These highly fluorescent fibrinogen conjugates are useful for investigating platelet activation
and subsequent fibrinogen binding using fluorescence microscopy or flow cytometry 112,115,118
(Figure 16.1.14).
The
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Probes Handbook:
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A Guide
Guide to
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Fluorescent Probes
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IMPORTANT
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Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
DQ™ Ovalbumin: A Probe for Antigen
Processing and Presentation
Although antigen processing and presentation have been extensively studied, the exact sequence and detailed pathways for generating
antigenic peptides have yet to be elucidated. In general, the immunogenic protein is internalized by a macrophage, denatured, reduced and
proteolyzed, and then the resulting peptides associate with MHC class
II molecules that are expressed at the cell surface.119 Ovalbumin is efficiently processed through mannose receptor–mediated endocytosis
by antigen-presenting cells and is widely used for studying antigen
processing.120–122 DQ™ ovalbumin 123 (D12053), a self-quenched ovalbumin conjugate, is designed specifically for the study of macrophagemediated antigen processing in flow cytometry and microscopy assays.
Traditionally, fluorescein-labeled bovine serum albumin (FITCBSA) has been used as a fluorogenic protein antigen for studying the
real-time kinetics of antigen processing in live macrophages by flow
cytometry,124 two-photon fluorescence lifetime imaging microscopy
(FLIM) 125 and fluorescence polarization.124,126,127 FITC-ovalbumin has
been employed to study antigen uptake in HIV-1–infected monocytic
cells.128 The FITC-ovalbumin and FITC-BSA used in these experiments
were heavily labeled with fluorescein such that the intact conjugates
were relatively nonfluorescent due to self-quenching. Upon denaturation and proteolysis, however, these FITC conjugates became highly
fluorescent, allowing researchers to monitor intracellular trafficking
and the processing of ovalbumin and BSA in macrophages.
For studies of antigen processing and presentation, DQ™ ovalbumin
offers several advantages when compared with FITC-ovalbumin and
FITC-BSA. Like the FITC conjugates, DQ™ ovalbumin is labeled with
our pH-insensitive, green-fluorescent BODIPY® FL dye such that the
fluorescence is almost completely quenched until the probe is digested
by proteases (Figure 16.1.15). Unlike fluorescein, which has greatly reduced fluorescence intensity at acidic pH and is not very photostable,
our BODIPY® FL dye exhibits bright, relatively photostable and pH-insensitive fluorescence from pH 3 to 9. Furthermore, the intact DQ™ ovalbumin is more highly quenched than unprocessed FITC-ovalbumin
or FITC-BSA at a lower degree of substitution, thereby providing a
lower background signal while preserving the protein’s antigenic epitopes. Although we offer the green-fluorescent DQ™ Green BSA and
red-fluorescent DQ™ Red BSA (D12050, D12051; Section 10.4), which
are also self-quenched BODIPY® FL and BODIPY® TR conjugates, we
highly recommend DQ™ ovalbumin (D12053) for studying antigen
processing and presentation 129,130 because ovalbumin is internalized
via the mannose receptor–mediated endocytosis pathway and is thus
processed more efficiently by antigen-presenting cells than is BSA.131
Section 16.1 Probes for Following Receptor Binding and Phagocytosis
gelatin and Oregon Green® 488 gelatin (G13187, G13186). Fluorescent
gelatin conjugates have been shown to be useful for:
• Assessing gelatinase activity in podosomes of mouse dendritic
cells 134
• Localizing surface fibronectin on cultured cells 135
• Performing in situ gelatinase zymography on canary brain
sections 136
• Studying fibronectin–gelatin interactions in solution using fluorescence polarization 133 (Fluorescence Polarization (FP)—Note 1.4)
We have also developed fluorogenic gelatinase and collagenase
substrates—DQ™ gelatin and DQ™ collagen (Figure 16.1.15) (D12054,
D12060)—that are described in Section 10.4. In addition, we offer fluorescent microspheres coated with collagen, which are described below.
Fluorescent Casein
Real-time imaging of fluorescein-labeled casein (C2990) and
FluoSpheres® fluorescent microspheres has been used to characterize the endocytic apparatus of the protozoan Giardia lamblia.137 The
EnzChek® Protease Assay Kits (E6638, E6639; Section 10.4) provide
convenient fluorescence-based assays for protease activity and contain either green-fluorescent BODIPY® FL casein or red-fluorescent
BODIPY® TR-X casein 138 (Figure 16.1.15). BODIPY® FL casein and
BODIPY® TR-X casein have significant utility as nontoxic and pHinsensitive general markers for phagocytic cells in culture.139,140 Our
RediPlate™ 96 (R22132) version of the BODIPY® TR-X casein substrate
(Section 10.4) is ideal for high-throughput screening of potential protease inhibitors.
Fluorescent Chemotactic Peptide
A variety of white blood cells containing the formyl-Met-Leu-Phe
(fMLF) receptor respond to bacterial N-formyl peptides by migrating to
the site of bacterial invasion and then initiating an activation pathway
to control the spread of infection. Activation involves Ca 2+ mobilization,141 transient acidification,142,143 actin polymerization,144 phagocytosis 145 and production of oxidative species.146 We offer the fluorescein
conjugate of the hexapeptide formyl-Nle-Leu-Phe-Nle-Tyr-Lys (F1314),
Enzyme
Fluorescent Gelatin
Collagen is a major component of the extracellular matrix and, in
vertebrates, constitutes approximately 25% of total protein. This important protein not only serves a structural role, but also is important
in cell adhesion and migration. Specific collagen receptors, fibronectin
and a number of other proteins involved in cell–cell and cell–surface
adhesion have been demonstrated to bind collagen and gelatin 132,133
(denatured collagen).
We offer highly fluorescent gelatin conjugates for researchers studying collagen-binding proteins and collagen metabolism, as well as gelatinases and collagenases, which are metalloproteins that digest gelatin and
collagen. We offer two green-fluorescent gelatin conjugates—fluorescein
Intramolecularly
quenched substrate
Fluorescent cleavage
products
Figure 16.1.15 Principle of enzyme detection via the disruption of intramolecular selfquenching. Enzyme-catalyzed hydrolysis of the heavily labeled and almost totally quenched
substrates provided in our EnzChek® Protease Assay Kits (E6638, E6639), EnzChek® Ultra
Amylase Assay Kit (E33651), EnzChek® Gelatinase/Collagenase Assay Kit (E12055), EnzChek®
Elastase Kit (E12056), EnzChek® Lysozyme Assay Kit (E22013)—as well as the stand-alone
quenched substrates DQ™ BSA (D12050, D12051), DQ™ collagen (D12052, D12060), DQ™ ovalbumin (D12053) and DQ™ gelatin (D12054)—relieves the intramolecular self-quenching,
yielding brightly fluorescent reaction products.
™
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.
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thermofisher.com/probes
747
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
Section 16.1 Probes for Following Receptor Binding and Phagocytosis
which has been extensively employed to investigate the fMLF receptor.147–151 The fluorescein-labeled chemotactic peptide has been used to study G-protein coupling and receptor structure,152–154
expression,155,156 distribution 157–159 and internalization.160
Fluorescent Insulin
Figure 16.1.16 Dexamethasone fluorescein (D1383).
A
We prepare a high-purity, zinc-free fluorescein isothiocyanate conjugate of human insulin
(FITC insulin, I13269). Unlike most commercially available preparations, our FITC insulin is purified by HPLC and consists of a singly labeled species of insulin that has been specifically modified
at the N-terminus of the B-chain. Because the degree and position of labeling can alter the biological activity of insulin, we have isolated the singly labeled species that has been shown to retain its
biological activity in an autophosphorylation assay.161 Our FITC insulin preparation is useful for
imaging insulin and insulin receptor distribution,162 as well as for conducting insulin-binding assays using microfluidic devices.163,164
Fluorescent Dexamethasone Probe for Glucocorticoid Receptors
B
The synthetic steroid hormone dexamethasone binds to the glucocorticoid receptor, producing a steroid–receptor complex that then localizes in the nucleus and regulates gene transcription.
In hepatoma tissue culture (HTC) cells, tetramethylrhodamine-labeled dexamethasone has been
shown to have high affinity for the glucocorticoid receptor in a cell-free system and to induce tyrosine aminotransferase (TAT) expression in whole cells, albeit at a much lower rate than unmodified
dexamethasone.165 This labeled dexamethasone also allowed the first observations of the fluorescent
steroid–receptor complex in the HTC cell cytosol.165 Fluorescein dexamethasone (D1383, Figure
16.1.16) should be similarly useful for studying the mechanism of glucocorticoid receptor activation.
Trypan blue
Fluorescent Histone H1
Figure 16.1.17 Principle of the Vybrant® Phagocytosis
Assay Kit (V6694) for the simple quantitation of phagocytosis. A) Briefly, phagocytic cells are incubated with the greenfluorescent fluorescein-labeled Escherichia coli BioParticles®
conjugates (E2861). B) The fluorescence from any noninternalized BioParticles® product is then quenched by the addition of trypan blue, and the samples are subsequently assayed with a fluorescence microplate reader equipped with
filters for the detection of fluorescein (FITC).
Normalized fluorescence
3.0
IC50 = 4.7 µM
The Alexa Fluor® 488 conjugate of the lysine-rich calf thymus histone H1 (H13188) is a useful
probe for nuclear protein transport assays.166 Nuclear-to-mitochondrial translocation of histone
H1 is indicative of dsDNA strand breaks. Fluorescent histone H1 conjugates can also be used to
detect membrane-surface exposure of acidic phospholipids such as phosphatidylserine.167
Fluorescent Probes for the Acrosome Reaction
Soybean trypsin inhibitor (SBTI) inhibits the catalytic activity of serine proteases and binds
to acrosin, an acrosomal serine protease associated with binding of spermatozoa to the zona
pellucida.168 Alexa Fluor® 488 dye–labeled trypsin inhibitor from soybean (T23011) is useful for
real-time imaging of the acrosome reaction in live spermatozoa.169 A fluorescent peanut lectin
has been combined with ethidium homodimer-1 (EthD-1, E1169; Section 15.2) for a combined
acrosome reaction assay and vital staining.170 Alexa Fluor® 488, Alexa Fluor® 568, Alexa Fluor®
594 and Alexa Fluor® 647 conjugates of Arachis hypogaea lectin (PNA) (L21409, L32458, L32459,
L32460) have similar utility as acrosomal stains.171
2.5
Methods for Detecting Internalized Fluorescent Ligands
2.0
1.5
1.0
0.5
10–9
10–8
10–7
10–6
10–5
10–4
[Dynasore] (M)
Figure 16.1.18 Tracking endocytosis inhibition with
pHrodo™ dextran conjugates. HeLa cells were plated in 96well format and treated with dynasore for 3 hours at 37°C
prior to the pHrodo™ endocytosis assay. Next, 40 µg/mL
of pHrodo™ 10,000 MW dextran (P10361) was incubated
for 30 minutes at 37°C, and cells were then stained with
HCS NuclearMask™ Blue Stain (H10325) for 10 minutes to
reveal total cell number and demarcation for image analysis. Images were acquired on the BD Pathway™ 855 HighContent Bioimager (BD Biosciences).
Many of the fluorescent ligands described in this section first bind to cell-surface receptors,
then are internalized and, in some cases, recycled to the cell surface. In most applications, the
cell-surface and internalized ligand populations are spatially resolved by imaging. It is often
desirable to include noninternalized plasma membrane reference markers in these labeling protocols. CellMask™ Orange and CellMask™ Deep Red plasma membrane stains (C10045, C10046;
Section 14.4) are particularly suitable for this purpose.172,173 Other useful membrane markers include posttranslationally lipidated fluorescent proteins 174 (O36214, O10139; Section 14.4). When
spatial resolution is not possible, there are other means by which these signals can be separated
and, in some cases, quantitated. These include:
• Use of antibodies to the Alexa Fluor® 488, BODIPY® FL, fluorescein/Oregon Green®,
tetramethylrhodamine, Texas Red® and Alexa Fluor® 405/Cascade Blue® dyes (Section 7.4,
Table 7.8) to quench most of the fluorescence of surface-bound or exocytosed probes
TheMolecular
MolecularProbes®
Probes Handbook:
Handbook: AAGuide
and Labeling
LabelingTechnologies
Technologies
The
Guideto
toFluorescent
Fluorescent Probes
Probes and
™
748
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 16 — Probes for Endocytosis, Receptors and Ion Channels
Section 16.1 Probes for Following Receptor Binding and Phagocytosis
• Use of a dye such as trypan blue to quench external fluorescent signals but not internalized
signals 175,176 (Figure 16.1.17)—a method employed in our Vybrant® Phagocytosis Assay Kit
(V6694) described below
• Rapid acidification of the medium to quench the fluorescence of pH-sensitive fluorophores
such as fluorescein on the cell surface, thus enabling selective detection of endocytosed probe
• Tagging of proteins, polysaccharides, cells, bacteria, yeast, fungi 177 and other materials to be
endocytosed with a pH-sensitive dye—such as our pHrodo™,178–180 SNARF® or Oregon Green®
dyes (Chapter 20)—that undergoes a spectral shift or intensity change in the acidic pH range
found in phagovacuoles and late endosomes
• Use of heavily labeled, highly quenched proteins such as our DQ™ BSA and DQ™ gelatin
probes, which yield highly fluorescent peptides upon intracellular proteolysis 181 (Section 10.4)
Pathway-specific inhibitors—such as chloropromazine, dynasore (Figure 16.1.18), dansyl
cadaverine (D113, Section 3.4), brefeldin A (B7450, Section 12.4), genistein and filipin—are
widely used in combination with fluorescently labeled ligands for characterizing endocytic
pathways.182 A critical evaluation 183 highlights some necessary cautions in the application and
interpretation of this approach, relating to decreased cell viability caused by some inhibitors as
well as cell type–dependent differences in their efficacy.
Figure 16.1.19 Live nerve terminals of motor neurons that
innervate a rat lumbrical muscle stained with the activitydependent dye FM® 1-43 (T3163, T35356) and observed
under low magnification. The dye molecules, which insert
into the outer leaflet of the surface membrane, are captured in recycled synaptic vesicles of actively firing neurons.
The image was contributed by William J. Betz, University of
Colorado School of Medicine.
Membrane Markers of Endocytosis and Exocytosis
FM® 1-43
FM® dyes—FM® 1-43, FM® 2-10, FM® 4-64, FM® 5-95 and the aldehyde-fixable FM® 1-43FX
and FM® 4-64FX—are excellent membrane probes both for identifying actively firing neurons 184
and for investigating the mechanisms of activity-dependent vesicle cycling in widely different
species.185–188 FM® dyes may also be useful as general-purpose probes for investigating endocytosis and for simply identifying cell membrane boundaries.
FM® 1-43 and its analogs, which are nontoxic to cells and virtually nonfluorescent in aqueous medium, are believed to insert into the outer leaflet of the surface membrane, where they
become intensely fluorescent. In a neuron that is actively releasing neurotransmitters, these
dyes become internalized within the recycled synaptic vesicles and the nerve terminals become
brightly stained (Figure 16.1.19, Figure 16.1.20). The nonspecific staining of cell-surface membranes can simply be washed off prior to viewing. Wash removal of noninternalized dye background is more difficult in tissue preparations than in disseminated cell cultures. Extracellular
fluorescence quenching 189 and dye adsorption 190 strategies have been developed to address this
problem. Alternatively, the optical sectioning capabilities of confocal microscopy, two-photon
excitation microscopy (Fluorescent Probes for Two-Photon Microscopy—Note 1.5) and total
internal reflection (TIRF) microscopy provide instrument-based solutions for improving the
signal-to-background contrast.191 The amount of FM® 1-43 taken up per vesicle by endocytosis
equals the amount of dye released upon exocytosis, indicating that the dye does not transfer
from internalized vesicles to an endosome-like compartment during the recycling process.192 In
astrocytes, internalization of FM® 1-43 (and FM® 4-64) is mediated by store-operated calcium
channels and not by endocytosis.193 Like most styryl dyes, the absorption and fluorescence
emission spectra of FM® 1-43 are significantly shifted in the membrane environment and are
relatively broad (Figure 16.1.21), requiring careful matching with other fluorophores to avoid
channel crosstalk in multiplex detection applications (Using the Fluorescence SpectraViewer—
Note 23.1). We offer FM® 1-43 in a 1 mg vial (T3163) or specially packaged in 10 vials of 100 µg
each (T35356).
FM® 1-43 was employed in a study showing that synaptosomal endocytosis is independent
of both extracellular Ca 2+ and membrane potential in dissociated hippocampal neurons,194 as
well as in a spectrofluorometric assay demonstrating that nitric oxide–stimulated vesicle release is independent of Ca 2+ in isolated rat hippocampal nerve terminals.195 FM® 1-43 has been
used in combination with fura-2 (Section 19.2) to simultaneously measure intracellular Ca 2+ and
membrane turnover.196,197 FM® 1-43 dye–mediated photoconversion has been used to visualize
recycling vesicles in hippocampal neurons.198
Figure 16.1.20 A feline mesenteric Pacinian corpuscle labeled with FM® 1-43 (T3163, T35356). The image was contributed by Michael Chua, University of North Carolina at
Chapel Hill.
Figure 16.1.21 Absorption and fluorescence emission spectra of FM® 1-43 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.
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thermofisher.com/probes
749
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
Section 16.1 Probes for Following Receptor Binding and Phagocytosis
Other Analogs of FM® 1-43
Figure 16.1.22 Feline muscle spindle, a specialized sensory receptor unit that detects muscle length and changes
in muscle length and velocity, was labeled with FM® 2-10
(T7508). Image contributed by Michael Chua, University of
North Carolina at Chapel Hill.
Figure 16.1.23 Correlated fluorescence imaging of membrane migration, protein translocation and chromosome
localization during Bacillus subtilis sporulation. Membranes
were stained with red-fluorescent FM® 4-64 (T3166, T13320).
Chromosomes were localized with the blue-fluorescent nuclear counterstain DAPI (D1306, D3571, D21490). The small,
green-fluorescent patches (top row) indicate the localization
of a GFP fusion to SpoIIIE, a protein essential for both initial
membrane fusion and forespore engulfment. Progression of
the engulfment is shown from left to right. Green fluorescence
in the middle and bottom rows demonstrates fully engulfed
sporangia stained with MitoTracker® Green FM® (M7514). Full
details of the experimental methods and interpretation are
published in Proc Natl Acad Sci U S A 96, 14553 (1999). Image
contributed by Kit Pogliano and Marc Sharp, University of
California at San Diego. Reproduced from the 7 December
1999 issue of Proc Natl Acad Sci U S A, with permission.
CH�
H ���(CH 2)� ��(CH 2)���
CH�
CH CH
��(CH2)�CH��2
��C�
Figure 16.1.24 FM® 1-43FX (F35355).
A comparison of mammalian motor nerve terminals stained with either FM® 1-43 or the
more hydrophilic analog FM® 2-10 (T7508, Figure 16.1.22) revealed that lower background staining by FM® 2-10 and its faster destaining rate may make it the preferred probe for quantitative
applications.199,200 However, staining with FM® 2-10 requires much higher dye concentrations 199
(100 µM compared with 2 µM for FM® 1-43). Additionally, it has been shown that both FM®
1-43 and FM® 2-10 are antagonists of muscarinic acetylcholine receptors and may be useful
for analyzing receptor distribution and occupancy.201 This property may be due to the cationic
alkylammonium substituent of FM® dyes, which they have in common with choline, and could
serve as one of the sources of background FM® dye staining in tissues.
FM® 4-64 (T3166, T13320) and RH 414 (T1111)—both more hydrophobic than FM® 1-43—
may also be useful as probes for investigating endocytosis. Because small differences in the polarity of these FM® probes can play a large role in their rates of uptake and their retention properties,
we have introduced FM® 5-95 (T23360), a slightly less lipophilic analog of FM® 4-64 with essentially identical spectroscopic properties. FM® 4-64 exhibits long-wavelength red fluorescence
that can be distinguished from Green Fluorescent Protein (GFP) with the proper optical filter
sets.202–205
FM® 4-64 is an endosomal marker and vital stain that persists through cell division, 206,207
as well as a stain for functional presynaptic boutons.208 In addition, FM® 4-64 staining has been
used to visualize membrane migration and fusion during Bacillus subtilis sporulation, and these
movements can be correlated with the translocation of GFP-labeled proteins 202,209,210 (Figure
16.1.23). Sequential pulse-chase application of FM® 4-64 and FM® 1-43 allows two-color fluorescence discrimination of temporally staged synaptic vesicle populations.187 FM® 4-64 selectively
stains yeast vacuolar membranes and is an important tool for visualizing vacuolar organelle morphology and dynamics and for studying the endocytic pathway and vacuole fusion in yeast 211–213
(Section 12.3). FM® 4-64 and FM® 1-43 also have many applications for visualizing membrane
dynamics in plant 204,214–216 and algal 217 cells.
FM® 1-43FX and FM® 4-64FX: Fixable FM® Dyes
FM® 1-43FX and FM® 4-64FX are FM® 1-43 and FM® 4-64 analogs, respectively, that have
been modified to contain an aliphatic amine (Figure 16.1.24, Figure 16.1.25). This modification
makes the probe fixable with aldehyde-based fixatives, including formaldehyde and glutaraldehyde. FM® 1-43FX has been used to study synaptic vesicle cycling in cone photoreceptor terminals 187 and to investigate the functional maturation of glutamatergic synapses.218 FM® 1-43FX
(F35355) and FM® 4-64FX (F34653) are available specially packaged in 10 vials of 100 µg each.
4-Di-1-ASP and 4-Di-2-ASP
The cationic mitochondrial dyes 4-Di-1-ASP (D288) and 4-Di-2-ASP (D289) stain presynaptic nerve terminals independent of neuronal activity.219–222 These aminostyrylpyridinium dyes
have also been widely used as substrates for functional analysis of biogenic amine transporters 223–227 and renal and hepatic organic cation transporters.228–230
TMA-DPH
CH �
H���(CH2)���(CH2)��
(CH
CH)�
�(CH2CH�)2
CH �
��C��C��
Figure 16.1.25 FM® 4-64FX (F34653).
Also useful as a lipid marker for endocytosis and exocytosis is the cationic linear polyene
TMA-DPH (T204, Figure 16.1.26), which readily incorporates in the plasma membrane of live
cells.231,232 TMA-DPH is virtually nonfluorescent in water and is reported to bind to cells in proportion to the available membrane surface.233 Its fluorescence intensity is therefore sensitive to
physiological processes that cause a net change in membrane surface area, making it an excellent
probe for monitoring events such as changes in cell volume and exocytosis.233–236
Fluorescent Cholera Toxin Subunit B: Markers of Lipid Rafts
Figure
16.1.26 TMA-DPH
(1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate)(T204).
Fluorescent cholera toxins, which bind to galactosyl moieties, are markers of lipid rafts—
regions of cell membranes high in ganglioside GM1 that are thought to be important in cell
signaling.237,238 Lipid rafts are detergent-insoluble, sphingolipid- and cholesterol-rich membrane microdomains that form lateral assemblies in the plasma membrane.239–245 Lipid rafts
also sequester glycophosphatidylinositol (GPI)-linked proteins and other signaling proteins and
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onto
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or more
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refer
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Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
Section 16.1 Probes for Following Receptor Binding and Phagocytosis
receptors, which may be regulated by their selective interactions with these membrane microdomains.246–251 Recent research has demonstrated that lipid rafts play a role in a variety of cellular
processes—including the compartmentalization of cell-signaling events, 252–259 the regulation of
apoptosis 260–262 and the intracellular trafficking of certain membrane proteins and lipids 263–265—
as well as in the infectious cycles of several viruses and bacterial pathogens.266–271
The Vybrant® Lipid Raft Labeling Kits (V34403, V34404, V34405; Section 14.4) provide the
key reagents for fluorescently labeling lipid rafts in vivo with our bright and extremely photostable Alexa Fluor® dyes (Figure 16.1.27, Figure 16.1.28). 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.250,272,273 An antibody that specifically recognizes CT-B is then used to crosslink the CT-B–
labeled lipid rafts into distinct patches on the plasma membrane, which are easily visualized by
fluorescence microscopy.274,275 Each Vybrant® Lipid Raft Labeling Kit contains sufficient reagents
to label 50 live-cell samples, 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 protocol
Figure 16.1.27 Live J774 macrophage cells labeled with
BODIPY® FL C5-ganglioside GM1 and Alexa Fluor® 555 cholera toxin subunit B conjugate. Live J774 macrophage cells
labeled with BODIPY® FL C5-ganglioside GM1 (B13950) and
then with Alexa Fluor® 555 cholera toxin subunit B conjugate (C22843; also available as a component of V34404).
Cells were then treated with anti–CT-B antibody (a component of V34404) to induce crosslinking. Yellow fluorescence
indicates colocalization of the two dyes. Nuclei were stained
with the blue-fluorescent Hoechst 33342 dye (H1399,
H3570, H21492).
Cholera toxin subunit B and its conjugates are also established as superior tracers for retrograde labeling of neurons.276,277 Cholera toxin subunit B conjugates bind to the pentasaccharide
chain of ganglioside GM1 on neuronal cell surfaces and are actively taken up and transported;
alternatively, they can be injected by iontophoresis. Unlike the carbocyanine-based neuronal
tracers such as DiI (D282, D3911, V22885; Section 14.4), cholera toxin subunit B conjugates can
be used on tissue sections that will be fixed and frozen.278
All of our cholera toxin subunit B conjugates are prepared from recombinant cholera toxin
subunit B, which is completely free of the toxic subunit A, thus eliminating any concern for
toxicity or ADP-ribosylating activity. The Alexa Fluor® 488 (C22841, C34775), Alexa Fluor® 555
(C22843, C34776), Alexa Fluor® 594 (C22842, C34777) and Alexa Fluor® 647 (C34778) conjugates of cholera toxin subunit B combine this versatile tracer with the superior brightness of our
Alexa Fluor® dyes to provide sensitive and selective receptor labeling and neuronal tracing. We
also offer biotin-XX (C34779) and horseradish peroxidase (C34780) conjugates of cholera toxin
subunit B for use in combination with diaminobenzidine (DAB) oxidation, 279 tyramide signal
amplification (TSA) and Qdot® nanocrystal–streptavidin conjugates.280
Fluorescent Protein–Based Lipid Raft Markers
CellLight® plasma membrane expression vectors (C10606, C10607, C10608; Section 14.4)
generate cyan-, green- or red-autofluorescent proteins fused to a plasma membrane targeting sequence consisting of the 10 N-terminal amino acids of LcK tyrosine kinase (Lck10). These fusion
proteins are lipid raft markers with well established utility,90 providing alternatives to cholera
toxin B conjugates or BODIPY® FL C5-ganglioside GM191 (B13950, B34401; Section 13.3) with the
inherent advantages of long-lasting and titratable expression conferred by BacMam 2.0 vector
technology (BacMam Gene Delivery and Expression Technology—Note 11.1).
Fluorescent Protein–Based Synaptic Vesicle Markers
CellLight® Synaptophysin-GFP (C10609) and CellLight® Synaptophysin-RFP (C10610) are
valuable counterparts to FM® dyes for visualizing the distribution and density of presynaptic sites
in neurons both in vitro and in vivo. Synaptophysin is a synaptic vesicle membrane glycoprotein
that is involved in the biogenesis and fusion of synaptic vesicles but is not essential for neurotransmitter release. It is found in virtually all synaptically active neurons in the brain and spinal cord.
These CellLight® reagents incorporate all the customary advantages of BacMam 2.0 delivery technology including high transduction efficiency and long-lasting and titratable expression (BacMam
Gene Delivery and Expression Technology—Note 11.1).
Figure 16.1.28 A J774 mouse macrophage cell stained
with BODIPY® FL ganglioside GM1 (B13950) and Alexa Fluor®
555 dye–labeled cholera toxin subunit B. 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 33258 (H1398,
H3569, H21491).
™
The
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TheMolecular
Molecular
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A Guide
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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
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Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
Section 16.1 Probes for Following Receptor Binding and Phagocytosis
Anti–Synapsin I Antibody
Figure 16.1.29 Peripheral neurons in mouse intestinal
cryosections were labeled with rabbit anti–synapsin I antibody (A6442) and detected using Alexa Fluor® 488 goat
anti–rabbit IgG antibody (A11008). This tissue was counterstained with DAPI (D1306, D3571, D21490).
Synapsin I is an actin-binding protein that is localized exclusively to synaptic vesicles and
thus serves as an reliable marker for synapses in brain and other neuronal tissues.283 Synapsin I
inhibits neurotransmitter release, an effect that is abolished upon its phosphorylation by Ca 2+/
calmodulin–dependent protein kinase II 284 (CaM kinase II). Antibodies directed against
synapsin I have proven valuable in molecular and neurobiology research, for example, to estimate
synaptic density and to follow synaptogenesis.218,285
We offer a rabbit polyclonal anti–bovine synapsin I antibody as an affinity-purified IgG fraction (A6442). This antibody was isolated from rabbits immunized against bovine brain synapsin
I but is also active against human, rat and mouse forms of the antigen; it has little or no activity
against synapsin II. The affinity-purified rabbit polyclonal antibody was fractionated from the
serum using column chromatography in which bovine synapsin I was covalently bound to the
column matrix. Affinity-purified anti–synapsin I antibody is suitable for immunohistochemistry
(Figure 16.1.29), western blots, enzyme-linked immunosorbent assays and immunoprecipitations.
Our complete selection of antibodies can be found at www.invitrogen.com/handbook/antibodies.
High Molecular Weight Polar Markers
Fluorescence emission
550
Fluorescent Protein–Based Endosomal Markers
pH 10
pH 8
pH 7
pH 6
pH 5
pH 4
Ex = 540 nm
600
650
CellLight® Early Endosomes–GFP (O10104) and CellLight® Early Endosomes–RFP (O36231)
provide BacMam expression vectors encoding fusions of GFP or RFP with the small GTPase
Rab5a. Rab5a fusions with autofluorescent proteins are sensitive and precise early endosome
markers for real-time imaging of clathrin-mediated endocytosis in live cells. 276,286,287 These
CellLight® reagents incorporate all the customary advantages of BacMam 2.0 delivery technology, including high transduction efficiency and long-lasting and titratable expression (BacMam
Gene Delivery and Expression Technology—Note 11.1).
700
Wavelength (nm)
Figure 16.1.30 The pH sensitivity of pHrodo™ dextran.
pHrodo™ 10,000 MW dextran (P10361) was reconsitituted in
HEPES (20 mM)–buffered PBS and adjusted to pH values from
pH 4 to pH 10. The intensity of fluorescence emission increases with increasing acidity, particularly in the pH 5–8 range.
20 µm
20 µm
0 min
20 µm
20 µm
40 min
80 min
120 min
Figure 16.1.31 Time course of pHrodo™ E. coli BioParticles® (P35361) uptake by metastatic malignant melanoma cells. Cells
were imaged at 37°C in the continued presence of 100 µg/mL pHrodo™ BioParticles®. Uptake of pHrodo™ BioParticles® was
observable as early as 20 minutes and reached a plateau within 2 to 3 hours.
Table 16.3 BioParticles® fluorescent bacteria and yeast.
Label
(Abs/Em Maxima in nm)
Escherichia coli
(K-12 strain)
Staphylococcus aureus
(Wood strain without protein A)
Zymosan A
(Saccharomyces cerevisiae)
Fluorescein (494/518)
E2861
S2851
Z2841
Alexa Fluor® 488 (495/519)
E13231
S23371
Z23373
S2854
BODIPY® FL (505/513)
E2864
Tetramethylrhodamine (555/580)
E2862
pHrodo™ (560/585)
A10025, P35361
A10010
Alexa Fluor® 594 (590/617)
E23370
S23372
Texas Red® (595/615)
E2863
Unlabeled
Z23374
Z2843
S2859
Z2849
We also offer opsonizing reagents for enhancing the uptake of BioParticles® products. These reagents are derived from purified rabbit polyclonal IgG antibodies that are specific for the E.
coli (E2870), S. aureus (S2860) or zymosan (Z2850) particles. Reconstitution of the lyophilized opsonizing reagents requires only the addition of water, and one unit of opsonizing reagent is
sufficient to opsonize ~10 mg of the corresponding BioParticles® product.
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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.
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Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
BioParticles® Fluorescent Bacteria and Yeast
The BioParticles® product line consists of a series of fluorescently
labeled, heat- or chemically killed bacteria and yeast in a variety of
sizes, shapes and natural antigenicities. These fluorescent BioParticles®
products have been employed to study phagocytosis by fluorescence
microscopy,288,289 quantitative spectrofluorometry 290 and flow cytometry.288,291 We offer Escherichia coli (K-12 strain), Staphylococcus aureus
(Wood strain without protein A) and zymosan (Saccharomyces cerevisiae) BioParticles® products covalently labeled with a variety of fluorophores, including Alexa Fluor®, fluorescein, BODIPY® FL, tetramethylrhodamine, Texas Red® and pHrodo™ dyes (Table 16.3). Special care
has been taken to remove any free dye after conjugation. BioParticles®
products are freeze-dried and ready for reconstitution in a buffer of
choice and are supplied with a general protocol for measuring phagocytosis; we also offer opsonizing reagents for use with each particle type,
as described below.
Unlike the fluorescence of fluorescein-labeled BioParticles® bacteria
and yeast, which is strongly quenched in acidic environments, the fluorescence of the Alexa Fluor® 488, BODIPY® FL, tetramethylrhodamine
and Texas Red® BioParticles® conjugates is uniformly intense between
pH 3 and 10. This property is particularly useful for quantitating fluorescent bacteria and zymosan within acidic phagocytic vacuoles.
Fluorescent bacteria and yeast particles are proven tools for studying a variety of phagocytosis parameters. For example, they have been
used to:
• Detect the phagocytosis of yeast by murine peritoneal macrophage 292 and human neutrophils 290
• Determine the effects of different opsonization procedures on the
efficiency of phagocytosis of pathogenic bacteria 293 and yeast 290
• Investigate the kinetics of phagocytosis degranulation and actin
polymerization in stimulated leukocytes 290
• Quantitate the effects of purinergic P2X7 receptor activation on
phagosomal maturation 179
• Show that Dictyostelium discoideum depleted of clathrin heavy
chains are still able to undergo phagocytosis of fluorescent
zymosans 294
• Study molecular defects in phagocytic function 178
Section 16.1 Probes for Following Receptor Binding and Phagocytosis
pHrodo™ BioParticles® Fluorescent Bacteria
In contrast to both the fluorescein- and Alexa Fluor® dye–labeled
BioParticles® conjugates, the fluorescence of the pHrodo™ E. coli and
S. aureus BioParticles® conjugates (P35361, A10010) increases in acidic
environments (Figure 16.1.30), providing a continuous positive indicator of phagocytic uptake. With a simple no-cell background subtraction
method, a large and specific signal is obtained from cells that ingest the
pHrodo™ BioParticles®, providing a specific index of phagocytosis in the
context of a variety of pretreatments or conditions (Figure 16.1.31). The
optimal absorption and fluorescence emission maxima of the pHrodo™
BioParticles® conjugates are approximately 560 nm and 585 nm, respectively, but the pHrodo™ fluorophore is also readily excited by the 488 nm
spectral line of the argon-ion laser used in most flow cytometers.
With each pHrodo™ BioParticles® conjugate, we provide sufficient
reagent for 100 microplate wells in a 96-well format, along with step-bystep instructions for performing phagocytosis assays in a fluorescence
microplate reader. This methodology has been developed using adherent J774A.1 murine macrophage cells, but can be adapted for use with
other adherent cells,178 primary cells 179,297 or cells in suspension,298 as
well as for in vivo applications.299 Cells assayed for phagocytic activity with pHrodo™ BioParticles® conjugates may be fixed with standard
formaldehyde solutions for later analysis, preserving differences in
signal between control and experimental samples with high fidelity.
pHrodo™ BioParticles® conjugate preparations are also amenable to opsonization (E2870, S2860), which can greatly enhance their uptake and
signal strength in the phagocytosis assay.
To facilitate the use of pHrodo™ BioParticles® conjugates for the
study of phagocytosis, we offer the pHrodo™ E. coli BioParticles®
Phagocytosis Kit for Flow Cytometry (A10025), which provides the key
reagents for assessing particle ingestion and red blood cell lysis (Figure
16.1.32). Each kit provides sufficient reagents for performing 100 assays
when using sample volumes of 100 µL whole blood per assay, including:
• pHrodo™ E. coli BioParticles® conjugates
• Lysis and wash buffers
• Detailed protocols
1,000
BioParticles® fluorescein-labeled Escherichia coli
Hanks’ balanced salt solution (HBSS)
Trypan blue
Step-by-step instructions for performing the phagocytosis assay
Number of cells counted
800
Side scatter
The Vybrant® Phagocytosis Assay Kit (V6694) provides a convenient set of reagents for quantitating phagocytosis and assessing the
effects of certain drugs or conditions on this cellular process. In this
assay, cells of interest are incubated first with green-fluorescent fluorescein-labeled E. coli BioParticles® conjugates, which are internalized
by phagocytosis, and then with trypan blue, which quenches the fluorescence of any extracellular BioParticles® product (Figure 16.1.17). The
methodology provided by this kit was developed using the adherent
murine macrophage cell line J774; 176 however, researchers have adapted
this assay to other phagocytic cell types 295 and other instrument platforms such as flow cytometers.296 Each kit provides sufficient reagents
for 250 tests using a 96-well microplate format and contains:
•
•
•
•
120
A
Vybrant® Phagocytosis Assay Kit
granulocytes
600
400
monocytes
200
debris
0
lymphocytes
0
200
400
600
Forward scatter
800
1,000
B
negative
control
80
phagocytosed
particles
40
0
100
101
102
103
104
pHrodo™ dye fluorescence (585 nm)
Figure 16.1.32 Flow cytometry analysis showing increased fluorescence of granulocytes
treated with pHrodo™ E. coli BioParticles® (P35361). A whole blood sample was collected and
treated with heparin, and two 100 µL aliquots were prepared. Both aliquots were treated
with pHrodo™ BioParticles® and vortexed. One sample was placed in a 37°C water bath, and
the other sample (negative control) was placed in an ice bath. After a 15-minute incubation,
red blood cells were lysed with an ammonium chloride–based lysis buffer. The samples were
centrifuged for 5 minutes at 500 rcf, washed once, and resuspended with HBSS. The samples
were then analyzed on a BD FACSCalibur™ cytometer (BD Biosciences) using a 488 nm argon
laser and 564–606 nm emission filter. A) Granulocytes were gated using forward and side
scatter. B) The sample incubated at 37°C shows the increased fluorescence of the phagocytosed pHrodo™ BioParticles® (red), in contrast to the negative control sample, which was kept
on ice to inhibit phagocytosis (blue).
™
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.
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753
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
Dextran
Oregon Green® 514
streptavidin
A
pHrodo™ Phagocytosis Particle Labeling Kit
In addition to the pHrodo™ BioParticles® conjugates, we offer the pHrodo™ Phagocytosis
Particle Labeling Kit for Flow Cytometry (A10026), which allows rapid labeling of biological
particles, such as bacteria, and subsequent assessment of of phagocytic activity in whole blood
samples by flow cytometry. Each kit provides sufficient reagents for performing 100 assays when
using sample volumes of 100 µL whole blood per assay, including:
•
•
•
•
•
B
Biotinylated
protein
Section 16.1 Probes for Following Receptor Binding and Phagocytosis
pHrodo™ succinimidyl ester
Lysis and wash buffers
Dimethylsulfoxide (DMSO)
Sodium bicarbonate
Detailed protocols
The amine-reactive pHrodo™ succinimidyl ester is also available separately (P36600, Section
20.4) for creating pH-sensitive conjugates for following phagocytosis. pHrodo™ succinimidyl ester was used to label dexamethasone-treated thymocytes for flow cytometry detection of phagocytosis by splenic or peritoneal macrophages.180
Opsonizing Reagents and Nonfluorescent BioParticles® Products
C
Many researchers may want to use autologous serum to opsonize their fluorescent zymosan
and bacterial particles; however, we also offer special opsonizing reagents (E2870, S2860, Z2850)
for enhancing the uptake of each type of particle, along with a protocol for opsonization. These
reagents are derived from purified rabbit polyclonal IgG antibodies that are specific for the E. coli,
S. aureus or zymosan particles. Reconstitution of the lyophilized opsonizing reagents requires
only the addition of water, and one unit of opsonizing reagent is sufficient to opsonize ~10 mg of
the corresponding BioParticles® product.
In addition, we offer nonfluorescent zymosan (Z2849) and S. aureus (S2859) BioParticles®
products. These nonfluorescent BioParticles® products are useful either as controls or for custom
labeling with the reactive dye or indicator of interest.
Endosomal
fusion
Fluorescent Polystyrene Microspheres
Figure 16.1.33 Detection of endosomal fusion. A) Cells
are first incubated with a combination of a high molecular
weight, red-fluorescent dextran (D1829, D1830, D1864)
and the green-fluorescent Oregon Green® 514 streptavidin
(S6369), which intrinsically has low fluorescence. B) The
cells are then incubated with a biotinylated probe, e.g., biotinylated transferrin (T23363), and the excess conjugate is
washed. C) Endosomal fusion is monitored by an increase in
fluorescence of the Oregon Green® 514 dye as it is displaced
by the biotinylated protein. The red-fluorescent dextran’s
fluorescence remains constant and allows for ratiometric
measurements of the fused endosomes.
Fluorescent polystyrene microspheres with diameters between 0.5 and 2.0 µm have been
used to investigate phagocytic processes in murine melanoma cells, 300 human alveolar macrophages,289 ciliated protozoa 137 and Dictyostelium discoideum.301,302 The phagocytosis of fluorescent microspheres has been quantitated both with image analysis 289,303,304 and with flow cytometry.305 Section 6.5 includes a detailed description of our full line of FluoSpheres® (Table 6.7)
and TransFluoSpheres® (Table 6.9) fluorescent microspheres. Because of their low nonspecific
binding, carboxylate-modified microspheres appear to be best for phagocytosis applications.
For phagocytosis experiments involving multicolor detection, we particularly recommend our
1.0 µm TransFluoSpheres® fluorescent microspheres 306 (T8880, T8883; Section 6.5). Various opsonizing reagents, such as rabbit serum or fetal calf serum, have been used with the microspheres
to facilitate phagocytosis.
Fluorescent Microspheres Coated with Collagen
Fibroblasts phagocytose and subsequently digest collagen. These activities play an important
role in the remodeling of the extracellular matrix during normal physiological turnover of connective tissues and wound repair, as well as in development and aging. A well-established procedure for observing collagen phagocytosis by either flow cytometry or fluorescence microscopy
entails the use of collagen-coated fluorescent microspheres that attach to the cell surface and
become engulfed by fibroblasts.307 We offer yellow-green–fluorescent FluoSpheres® collagen I–
labeled microspheres in either 1.0 µm or 2.0 µm diameter (F20892, F20893) for use in these applications. In the production of these microspheres, collagen I from calf skin is attached covalently
to the microsphere’s surface.
TheMolecular
MolecularProbes®
Probes Handbook:
Handbook: AAGuide
and Labeling
Labeling Technologies
Technologies
The
GuidetotoFluorescent
Fluorescent Probes
Probes and
™
754
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.
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thermofisher.com/probes
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
Section 16.1 Probes for Following Receptor Binding and Phagocytosis
Fluorescent Dextrans
Em = 580 nm
Fluorescence excitation
Tracing internalization of extracellularly introduced fluorescent dextrans is a standard
method for analyzing fluid-phase endocytosis.2,73,308,309 We offer dextrans with nominal molecular weights ranging from 3000 to 2,000,000 daltons, many of which can also be used as
pinocytosis or phagocytosis markers (see Section 14.5 and Table 14.4 for further discussion and
a complete product list). Discrimination of internalized fluorescent dextrans from dextrans in
the growth medium is facilitated by use of reagents that quench the fluorescence of the external
probe. For example, most of our anti-fluorophore antibodies (Section 7.4, Table 7.8) strongly
quench the fluorescence of the corresponding dyes.
Negative staining produced by fluorescent dextrans that have been intracellularly infused
via a patch pipette is indicative of nonendocytic vacuoles in live pancreatic acinar cells.310
Extracellular addition of a second, color-contrasting dextran then allows discrimination of endocytic and nonendocytic vacuoles. Intracellular fusion of endosomes has been monitored with
a BODIPY® FL avidin conjugate by following the fluorescence enhancement that occurs when
it complexes with a biotinylated dextran.311 We have found our Oregon Green® 514 streptavidin
(S6369, Section 7.6) to have an over 15-fold increase in fluorescence intensity upon binding free
biotin, which may make it the preferred probe for this application (Figure 16.1.33).
pH 8.0
7.0
6.6
6.3
6.0
5.5
5.0
450
500
550
600
Wavelength (nm)
Figure 16.1.34 The excitation spectra of double-labeled
fluorescein-tetramethylrhodamine dextran (D1951), which
contains pH-dependent (fluorescein) and pH-independent
(tetramethylrhodamine) dyes.
pH Indicator Dextrans
The fluorescein dextrans (pKa ~6.4) are frequently used to investigate endocytic acidification.312,313 Fluorescence of fluorescein-labeled dextrans is strongly quenched upon acidification;
however, fluorescein’s lack of a spectral shift in acidic solution makes it difficult to discriminate
between an internalized probe that is quenched and residual fluorescence of the external medium. Dextran conjugates that either shift their emission spectra in acidic environments, such
as the SNARF® dextrans (Section 20.4), or undergo significant shifts of their excitation spectra,
such as BCECF and Oregon Green® dextrans (Section 20.4), provide alternatives to fluorescein.
The Oregon Green® 488 and Oregon Green® 514 dextrans exhibit a pKa of approximately 4.7,
facilitating measurements in acidic environments.312,314 In addition to these pH indicator dextrans, we prepare a dextran that is double-labeled with fluorescein and tetramethylrhodamine
(D1951; Section 20.4), which has been used as a ratiometric indicator (Figure 16.1.34) to measure
endosomal acidification in Hep G2 cells 315 and murine alveolar macrophages.178
In contrast to fluorescein and Oregon Green® 488 dextrans, pHrodo™ 10,000 MW dextran
(P10361) exhibits increasing fluorescence in response to acidification 178 (Figure 16.1.30). The
minimal fluorescent signal from pHrodo™ dextran at neutral pH prevents the detection of noninternalized and nonspecifically bound conjugates and eliminates the need for quenching reagents
and extra wash steps, thus providing a simple fluorescent assay for endocytic activity. pHrodo™
dextran’s excitation and emission maxima of 560 and 585 nm, respectively, facilitate multiplexing with other fluorophores including blue-, green- and far-red–fluorescent probes. Although
pHrodo™ dextran is optimally excited at approximately 560 nm, it is also readily excited by the
488 nm spectral line of the argon-ion laser found on flow cytometers, confocal microscopes and
imaging microplate readers (Figure 16.1.18).
Low Molecular Weight Polar Markers
Hydrophilic fluorescent dyes—including sulforhodamine 101 (S359), lucifer yellow CH (L453),
calcein (C481), 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS, pyranine; H348) and Cascade Blue®
hydrazide (C687)—are taken up by actively firing neurons through endocytic recycling of the synaptic vesicles.316,317 Unlike the fluorescent FM® membrane probes described above, however, the
hydrophilic fluorophores appear to work for only a limited number of species in this application. In
some tissue preparations, background due to noninternalized polar markers is easier to wash away
than that emanating from membrane markers such as FM® 1-43.316 The same dyes have frequently
been used as fluid-phase markers of pinocytosis.318–321 The highly water-soluble Alexa Fluor® hydrazides and Alexa Fluor® hydroxylamines (Section 14.3, Table 3.2) provide superior spectral properties and can be fixed in cells by aldehyde-based fixatives.322
™
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
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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
755
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
Section 16.1 Probes for Following Receptor Binding and Phagocytosis
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The
MolecularProbes®
Probes Handbook:
Handbook: AAGuide
and Labeling
LabelingTechnologies
Technologies
The
Molecular
Guideto
toFluorescent
Fluorescent Probes
Probes and
™
756
IMPORTANT
NOTICE:
The products
described
in this manual
covered
by covered
one or more
Limited
Use Label
License(s).
Please
refer to thePlease
Appendix
onto
IMPORTANT
NOTICE
: The products
described
in thisaremanual
are
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 16 — Probes for Endocytosis, Receptors and Ion Channels
Section 16.1 Probes for Following Receptor Binding and Phagocytosis
DATA TABLE 16.1 PROBES FOR FOLLOWING RECEPTOR BINDING AND PHAGOCYTOSIS
Cat. No.
MW
Storage
Soluble
Abs
EC
Em
Solvent
Notes
C481
622.54
L
pH >5
494
77,000
517
pH 9
1
C687
596.44
L
H 2O
399
30,000
421
H2O
2, 3
D288
366.24
L
DMF
475
45,000
605
MeOH
4
D289
394.30
L
H2O, DMF
488
48,000
607
MeOH
4
D1383
840.98
L
pH >6, DMF
494
76,000
519
pH 9
D2935
584.37
F,D,AA
DMF
258
11,000
none
MeOH
5
E3476
~6100
FF,D
H 2O
<300
none
E3477
~6600
FF,D
H2O
<300
none
6
E3478
~6500
FF,D,L
H 2O
495
84,000
517
pH 8
6, 7
E3480
see Notes
FF,D,L
H 2O
596
ND
612
pH 7
8, 9
E3481
~6800
FF,D,L
H 2O
555
85,000
581
pH 7
6, 7
E7498
~6600
FF,D,L
H 2O
511
85,000
528
pH 9
6, 7
E13345
see Notes
FF,D,L
H 2O
497
ND
520
pH 8
8, 10
E35350
see Notes
FF,D,L
H 2O
554
ND
568
pH 7
8, 11
E35351
see Notes
FF,D,L
H 2O
653
ND
671
pH 7
8, 12
F1314
1213.41
F,L
pH >6, DMF
494
72,000
517
pH 9
F2902
see Notes
RR,L,AA
H 2O
<300
none
13, 14, 15
F34653
788.75
D,L
H2O, DMSO
562
47,000
744
CHCl3
4
F35355
560.09
D,L
H2O, DMSO
510
50,000
626
MeOH
4
H348
524.37
D,L
H 2O
454
24,000
511
pH 9
16
L453
457.24
L
H 2O
428
12,000
536
H2O
17, 18
L3482
see Notes
RR,L,AA
see Notes
554
ND
571
see Notes
8, 19, 20, 21
L3483
see Notes
RR,L,AA
see Notes
515
ND
520
see Notes
8, 19, 20, 21
L3484
see Notes
RR,L,AA
see Notes
554
ND
571
see Notes
8, 19, 20, 21
L3485
see Notes
RR,L,AA
see Notes
510
ND
518
see Notes
8, 19, 20, 21
L23380
see Notes
RR,L,AA
see Notes
495
ND
519
see Notes
8, 19, 20, 21
S359
606.71
L
H 2O
586
108,000
605
H2O
T204
461.62
D,L
DMF, DMSO
355
75,000
430
MeOH
22
T1111
581.48
D,L
DMSO, EtOH
532
55,000
716
MeOH
4, 23
T3163
611.55
D,L
H2O, DMSO
471
38,000
581
see Notes
24, 25
T3166
607.51
D,L
H2O, DMSO
505
47,000
725
see Notes
24, 26
T7508
555.44
D,L
H2O, DMSO
506
50,000
620
MeOH
4
T13320
607.51
D,L
H2O, DMSO
505
47,000
725
see Notes
24, 26
T23360
565.43
D,L
H2O, DMSO
560
43,000
734
CHCl3
26
T35356
611.55
D,L
H2O, DMSO
471
38,000
581
see Notes
24, 25
For definitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.
Notes
1. C481 fluorescence is strongly quenched by micromolar concentrations of Fe3+, Co2+, Ni2+ and Cu2+ at pH 7. (Am J Physiol (1995) 268:C1354, J Biol Chem (1999) 274:13375)
2. The Alexa Fluor® 405 and Cascade Blue® dyes have a second absorption peak at about 376 nm with EC ~80% of the 395–400 nm peak.
3. Maximum solubility in water is ~1% for C687, ~1% for C3221 and ~8% for C3239.
4. 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.
5. Dihydrofluorescein diacetates are colorless and nonfluorescent until both of the acetate groups are hydrolyzed and the products are subsequently oxidized to fluorescein derivatives. The materials contain less than 0.1% of oxidized derivative when initially prepared. The oxidation products of C400, C2938, C6827, D399 and D2935 are 2’,7’-dichlorofluorescein derivatives with spectra
similar to C368 (see data).
6. α-Bungarotoxin, EGF and phallotoxin conjugates have approximately 1 label per peptide.
7. The value of EC listed for this EGF conjugate is for the labeling dye in free solution. Use of this value for the conjugate assumes a 1:1 dye:peptide labeling ratio and no change of EC due to dye–
peptide interactions.
8. ND = not determined.
9. E3480 is a complex of E3477 with Texas Red® streptavidin, which typically incorporates 3 dyes/streptavidin (MW ~52,800).
10. E13345 is a complex of E3477 with Alexa Fluor® 488 streptavidin, which typically incorporates 5 dyes/streptavidin (MW ~52,800).
11. E35350 is a complex of E3477 with Alexa Fluor® 555 streptavidin, which typically incorporates 3 dyes/streptavidin (MW ~52,800).
12. E35351 is a complex of E3477 with Alexa Fluor® 647 streptavidin, which typically incorporates 3 dyes/streptavidin (MW ~52,800).
13. This product is supplied as a ready-made solution in the solvent indicated under “Soluble.”
14. F2902 is essentially colorless and nonfluorescent until oxidized. A small amount (~5%) of oxidized material is normal and acceptable for the product as supplied. The oxidation product is
fluorescent (Abs = 495 nm, Em = 524 nm). (J Immunol Methods (1990) 130:223)
15. This product consists of a dye–bovine serum albumin conjugate (MW ~66,000) complexed with IgG in a ratio of approximately 1:4 mol:mol (BSA:IgG)
16. H348 spectra are pH-dependent.
17. The fluorescence quantum yield of lucifer yellow CH in H2O is 0.21. (J Am Chem Soc (1981) 103:7615)
18. Maximum solubility in water is ~8% for L453, ~6% for L682 and ~1% for L1177.
19. LDL complexes must be stored refrigerated BUT NOT FROZEN. The maximum shelf-life under the indicated storage conditions is 4–6 weeks.
20. This LDL complex incorporates multiple fluorescent labels. The number of dyes per apoprotein B (MW ~500,000) is indicated on the product label.
21. LDL complexes are packaged under argon in 10 mM Tris, 150 mM NaCl, 0.3 mM EDTA, pH 8.3 containing 2 mM azide. Spectral data reported are measured in this buffer.
22. 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.
23. RH 414 Abs ~500 nm, Em ~635 nm when bound to phospholipid bilayer membranes.
24. Abs, EC and Em determined for dye bound to detergent micelles (20 mg/mL CHAPS in H2O). These dyes are essentially nonfluorescent in pure water.
25. FM® 1-43 Abs = 479 nm, Em = 598 nm bound to phospholipid bilayer membranes. Em = 565 nm bound to synaptosomal membranes. (Neuron (1994) 12:1235)
26. FM® 4-64 and FM® 5-95 are nonfluorescent in water. For two-color imaging in GFP-expressing cells, these dyes can be excited at 568 nm with emission detection at 690–730 nm. (Am J Physiol
Cell Physiol (2001) 281:C624)
™
The
Probes
Handbook:
A Guide
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Probes
and
Labeling
Technologies
TheMolecular
Molecular
Probes®
Handbook:
A Guide
to Fluorescent
Probes
and
Labeling
Technologies
IMPORTANT
NOTICE:described
The products
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by oneLimited
or moreUse
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Use
Label License(s).
to the
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IMPORTANT NOTICE
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in this
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Label
License(s).
PleasePlease
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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.
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Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
Section 16.1 Probes for Following Receptor Binding and Phagocytosis
PRODUCT LIST 16.1 PROBES FOR FOLLOWING RECEPTOR BINDING AND PHAGOCYTOSIS
Cat. No.
Product
A6442
A11130
C481
C687
C2990
C10586
C10587
C10609
C10610
C34775
C22841
C34776
C22843
C34777
C22842
C34778
C34779
C34780
D1383
D2935
D289
D288
D12060
D12054
D12050
D12053
D12051
E3476
E3477
E13345
E35350
E35351
E3480
E3478
E7498
E3481
E2870
E13231
E23370
E2864
E2861
E2862
E2863
F2902
F13191
F13192
F13193
F35200
F7496
F20892
F20893
F35355
F34653
F1314
G13187
G13186
H13188
H348
I13269
anti-synapsin I (bovine), rabbit IgG fraction *affinity purified*
anti-transferrin receptor (human), mouse IgG1, monoclonal 236-15375
calcein *high purity*
Cascade Blue® hydrazide, trisodium salt
casein, fluorescein conjugate
CellLight® Early Endosomes-GFP
CellLight® Early Endosomes-RFP
CellLight® Synaptophysin-GFP
CellLight® Synaptophysin-RFP
cholera toxin subunit B (recombinant), Alexa Fluor® 488 conjugate
cholera toxin subunit B (recombinant), Alexa Fluor® 488 conjugate
cholera toxin subunit B (recombinant), Alexa Fluor® 555 conjugate
cholera toxin subunit B (recombinant), Alexa Fluor® 555 conjugate
cholera toxin subunit B (recombinant), Alexa Fluor® 594 conjugate
cholera toxin subunit B (recombinant), Alexa Fluor® 594 conjugate
cholera toxin subunit B (recombinant), Alexa Fluor® 647 conjugate
cholera toxin subunit B (recombinant), biotin-XX conjugate
cholera toxin subunit B (recombinant), horseradish peroxidase conjugate
dexamethasone fluorescein
2’,7’-dichlorodihydrofluorescein diacetate, succinimidyl ester (OxyBURST® Green H2DCFDA, SE)
4-(4-(diethylamino)styryl)-N-methylpyridinium iodide (4-Di-2-ASP)
4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (4-Di-1-ASP)
DQ™ collagen, type I from bovine skin, fluorescein conjugate
DQ™ gelatin from pig skin, fluorescein conjugate *special packaging*
DQ™ Green BSA *special packaging*
DQ™ ovalbumin *special packaging*
DQ™ Red BSA *special packaging*
epidermal growth factor (EGF) *from mouse submaxillary glands*
epidermal growth factor, biotin-XX conjugate (biotin EGF)
epidermal growth factor, biotinylated, complexed to Alexa Fluor® 488 streptavidin (Alexa Fluor® 488 EGF complex)
epidermal growth factor, biotinylated, complexed to Alexa Fluor® 555 streptavidin (Alexa Fluor® 555 EGF complex)
epidermal growth factor, biotinylated, complexed to Alexa Fluor® 647 streptavidin (Alexa Fluor® 647 EGF complex)
epidermal growth factor, biotinylated, complexed to Texas Red® streptavidin (Texas Red® EGF complex)
epidermal growth factor, fluorescein conjugate (fluorescein EGF)
epidermal growth factor, Oregon Green® 514 conjugate (Oregon Green® 514 EGF)
epidermal growth factor, tetramethylrhodamine conjugate (rhodamine EGF)
Escherichia coli BioParticles® opsonizing reagent
Escherichia coli (K-12 strain) BioParticles®, Alexa Fluor® 488 conjugate
Escherichia coli (K-12 strain) BioParticles®, Alexa Fluor® 594 conjugate
Escherichia coli (K-12 strain) BioParticles®, BODIPY® FL conjugate
Escherichia coli (K-12 strain) BioParticles®, fluorescein conjugate
Escherichia coli (K-12 strain) BioParticles®, tetramethylrhodamine conjugate
Escherichia coli (K-12 strain) BioParticles®, Texas Red® conjugate
Fc OxyBURST® Green assay reagent *25 assays* *3 mg/mL*
fibrinogen from human plasma, Alexa Fluor® 488 conjugate
fibrinogen from human plasma, Alexa Fluor® 546 conjugate
fibrinogen from human plasma, Alexa Fluor® 594 conjugate
fibrinogen from human plasma, Alexa Fluor® 647 conjugate
fibrinogen from human plasma, Oregon Green® 488 conjugate
FluoSpheres® collagen I-labeled microspheres, 1.0 µm, yellow-green fluorescent (505/515) *0.5% solids*
FluoSpheres® collagen I-labeled microspheres, 2.0 µm, yellow-green fluorescent (505/515) *0.5% solids*
FM® 1-43FX *fixable analog of FM® 1-43 membrane stain*
FM® 4-64FX *fixable analog of FM® 4-64 membrane stain*
formyl-Nle-Leu-Phe-Nle-Tyr-Lys, fluorescein derivative
gelatin from pig skin, fluorescein conjugate
gelatin from pig skin, Oregon Green® 488 conjugate
histone H1 from calf thymus, Alexa Fluor® 488 conjugate
8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS; pyranine)
insulin, human, recombinant from E. coli, fluorescein conjugate (FITC insulin) *monolabeled* *zinc free*
Quantity
The
MolecularProbes®
Probes Handbook:
Handbook: AAGuide
Probesand
andLabeling
LabelingTechnologies
Technologies
The
Molecular
Guide to
to Fluorescent
Fluorescent Probes
™
758
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
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10 µg
50 µg
100 mg
10 mg
25 mg
1 kit
1 kit
1 kit
1 kit
100 µg
500 µg
100 µg
500 µg
100 µg
500 µg
100 µg
100 µg
100 µg
5 mg
5 mg
1g
1g
1 mg
5 x 1 mg
5 x 1 mg
5 x 1 mg
5 x 1 mg
100 µg
20 µg
100 µg
100 µg
100 µg
100 µg
20 µg
20 µg
20 µg
1U
2 mg
2 mg
10 mg
10 mg
10 mg
10 mg
500 µL
5 mg
5 mg
5 mg
5 mg
5 mg
0.4 mL
0.4 mL
10 x 100 µg
10 x 100 µg
1 mg
5 mg
5 mg
1 mg
1g
100 µg
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
Section 16.1 Probes for Following Receptor Binding and Phagocytosis
PRODUCT LIST 16.1 PROBES FOR FOLLOWING RECEPTOR BINDING AND PHAGOCYTOSIS—continued
Cat. No.
Product
L21409
L32458
L32459
L32460
L23351
L23352
L23353
L23350
L23356
L3486
L35354
L23380
L35353
L3485
L3484
L3483
L3482
L453
O13291
P10361
P35361
A10025
A10026
A10010
S2860
S23371
S23372
S2854
S2851
S2859
S359
T204
T13342
T23364
T35352
T23365
T13343
T23362
T23366
T35357
T23363
T2871
T2872
T2875
T3163
T35356
T1111
T3166
T13320
T7508
T23360
T23011
V6694
Z2850
Z23373
Z23374
Z2841
Z2843
Z2849
lectin PNA from Arachis hypogaea (peanut), Alexa Fluor® 488 conjugate
lectin PNA from Arachis hypogaea (peanut), Alexa Fluor® 568 conjugate
lectin PNA from Arachis hypogaea (peanut), Alexa Fluor® 594 conjugate
lectin PNA from Arachis hypogaea (peanut), Alexa Fluor® 647 conjugate
lipopolysaccharides from Escherichia coli serotype 055:B5, Alexa Fluor® 488 conjugate
lipopolysaccharides from Escherichia coli serotype 055:B5, Alexa Fluor® 568 conjugate
lipopolysaccharides from Escherichia coli serotype 055:B5, Alexa Fluor® 594 conjugate
lipopolysaccharides from Escherichia coli serotype 055:B5, BODIPY® FL conjugate
lipopolysaccharides from Salmonella minnesota, Alexa Fluor® 488 conjugate
low-density lipoprotein from human plasma (LDL) *2.5 mg/mL*
low-density lipoprotein from human plasma, acetylated (AcLDL) *2.5 mg/mL*
low-density lipoprotein from human plasma, acetylated, Alexa Fluor® 488 conjugate (Alexa Fluor® 488 AcLDL) *1 mg/mL*
low-density lipoprotein from human plasma, acetylated, Alexa Fluor® 594 conjugate (Alexa Fluor® 594 AcLDL) *1 mg/mL*
low-density lipoprotein from human plasma, acetylated, BODIPY® FL conjugate (BODIPY® FL AcLDL) *1 mg/mL*
low-density lipoprotein from human plasma, acetylated, DiI complex (DiI AcLDL) *1 mg/mL*
low-density lipoprotein from human plasma, BODIPY® FL complex (BODIPY® FL LDL) *1 mg/mL*
low-density lipoprotein from human plasma, DiI complex (DiI LDL) *1 mg/mL*
lucifer yellow CH, lithium salt
OxyBURST® Green H2HFF BSA *special packaging*
pHrodo™ dextran, 10,000 MW *for endocytosis*
pHrodo™ E. coli BioParticles® conjugate for phagocytosis
pHrodo™ E. coli BioParticles® Phagocytosis Kit *for flow cytometry* *100 tests*
pHrodo™ Phagocytosis Particle Labeling Kit *for flow cytometry* *100 tests*
pHrodo™ S. aureus BioParticles® conjugate for phagocytosis
Staphylococcus aureus BioParticles® opsonizing reagent
Staphylococcus aureus (Wood strain without protein A) BioParticles®, Alexa Fluor® 488 conjugate
Staphylococcus aureus (Wood strain without protein A) BioParticles®, Alexa Fluor® 594 conjugate
Staphylococcus aureus (Wood strain without protein A) BioParticles®, BODIPY® FL conjugate
Staphylococcus aureus (Wood strain without protein A) BioParticles®, fluorescein conjugate
Staphylococcus aureus (Wood strain without protein A) BioParticles®, unlabeled
sulforhodamine 101
TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate)
transferrin from human serum, Alexa Fluor® 488 conjugate
transferrin from human serum, Alexa Fluor® 546 conjugate
transferrin from human serum, Alexa Fluor® 555 conjugate
transferrin from human serum, Alexa Fluor® 568 conjugate
transferrin from human serum, Alexa Fluor® 594 conjugate
transferrin from human serum, Alexa Fluor® 633 conjugate
transferrin from human serum, Alexa Fluor® 647 conjugate
transferrin from human serum, Alexa Fluor® 680 conjugate
transferrin from human serum, biotin-XX conjugate
transferrin from human serum, fluorescein conjugate
transferrin from human serum, tetramethylrhodamine conjugate
transferrin from human serum, Texas Red® conjugate
N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM® 1-43)
N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM® 1-43) *special packaging*
N-(3-triethylammoniumpropyl)-4-(4-(4-(diethylamino)phenyl)butadienyl)pyridinium dibromide (RH 414)
N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM® 4-64)
N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM® 4-64) *special packaging*
N-(3-triethylammoniumpropyl)-4-(4-(diethylamino)styryl)pyridinium dibromide (FM® 2-10)
N-(3-trimethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM® 5-95)
trypsin inhibitor from soybean, Alexa Fluor® 488 conjugate
Vybrant® Phagocytosis Assay Kit *250 assays*
zymosan A BioParticles® opsonizing reagent
zymosan A (S. cerevisiae) BioParticles®, Alexa Fluor® 488 conjugate
zymosan A (S. cerevisiae) BioParticles®, Alexa Fluor® 594 conjugate
zymosan A (S. cerevisiae) BioParticles®, fluorescein conjugate
zymosan A (S. cerevisiae) BioParticles®, Texas Red® conjugate
zymosan A (S. cerevisiae) BioParticles®, unlabeled
Quantity
1 mg
1 mg
1 mg
1 mg
100 µg
100 µg
100 µg
100 µg
100 µg
200 µL
200 µL
200 µL
200 µL
200 µL
200 µL
200 µL
200 µL
25 mg
5 x 1 mg
0.5 mg
5 x 2 mg
1 kit
1 kit
5 x 2 mg
1U
2 mg
2 mg
10 mg
10 mg
100 mg
25 mg
25 mg
5 mg
5 mg
5 mg
5 mg
5 mg
5 mg
5 mg
5 mg
5 mg
5 mg
5 mg
5 mg
1 mg
10 x 100 µg
5 mg
1 mg
10 x 100 µg
5 mg
1 mg
1 mg
1 kit
1U
2 mg
2 mg
10 mg
10 mg
100 mg
™
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
759
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
Section 16.2 Probes for Neurotransmitter Receptors
16.2 Probes for Neurotransmitter Receptors
Fluorescent receptor ligands provide a sensitive means of identifying and localizing various cellular receptors, ion channels and ion
carriers. Many of these site-selective fluorescent probes may be used
on live or fixed cells, as well as in cell-free extracts. The high sensitivity
and selectivity of these fluorescent probes make them especially good
candidates for measuring low-abundance receptors.1–5 Various methods for further amplifying detection of these receptors 6,7 are discussed
in Chapter 6 and Chapter 7.
This section is devoted to our probes for neurotransmitter receptors. Additional fluorescently labeled receptor ligands (including lowdensity lipoproteins, epidermal growth factors, transferrin and fibrinogen conjugates and chemotactic peptides) are described in Section 16.1,
along with other probes for studying receptor-mediated endocytosis.
Section 16.3 describes a variety of probes for Ca 2+, Na+, K+ and Cl– ion
channels and carriers. Chapter 17 focuses on reagents for investigating
events—such as calcium regulation, kinase, phosphatase and phospholipase activation, and lipid trafficking—that occur downstream from
the receptor–ligand interaction (Figure 16.2.1).
α-Bungarotoxin Probes for Nicotinic
Acetylcholine Receptors
Fluorescent α-Bungarotoxins
Nicotinic acetylcholine receptors (nAChRs) are neurotransmittergated ion channels that produce an increase in Na+ and K+ permeability, depolarization and excitation upon activation by acetylcholine8
(Figure 16.2.1). α-Bungarotoxin is a 74–amino acid (~8000 dalton)
peptide containing 5 lysine residues and 10 cysteine residues paired
in 5 disulfide bridges. Extracted from Bungarus multicinctus venom,
α-bungarotoxin binds with high affinity to the α-subunit of the nAChR
of neuromuscular junctions.9 We provide an extensive selection of
fluorescent α-bungarotoxin conjugates (Table 16.4) to facilitate visualization of nAChRs with a variety of instrumentation. We attach approximately one fluorophore to each molecule of α-bungarotoxin, thus
retaining optimal binding specificity. The labeled bungarotoxins are
then chromatographically separated from unlabeled molecules to ensure adequate labeling of the product.
Alexa Fluor® 488 α-bungarotoxin (B13422) has fluorescence
spectra similar to those of fluorescein α-bungarotoxin (F1176) and is
therefore suitable for use with standard fluorescein optical filter sets.
Tetramethylrhodamine α-bungarotoxin 10–12 (T1175) has been the preferred red-orange–fluorescent probe for staining the nAChR (Figure
16.2.2). We not only offer the red-orange–fluorescent Alexa Fluor® 555
α-bungarotoxin (B35451), but also the red-fluorescent Alexa Fluor®
594 α-bungarotoxin (B13423), which has a longer-wavelength emission maximum and therefore offers better spectral separation from
green-fluorescent dyes in multicolor experiments. Our two longestwavelength conjugates—Alexa Fluor® 647 α-bungarotoxin (B35450)
and Alexa Fluor® 680 α-bungarotoxin (B35452)—are spectrally separated from both green-fluorescent and orange-fluorescent dyes, allowing researchers to easily perform three- and four-color experiments.
Fluorescent α-bungarotoxins have been used in a variety of informative investigations to:
• Correlate receptor clustering during neuromuscular development
with tyrosine phosphorylation of the receptor 13,14
• Detect reinnervation of adult muscle after nerve damage and to
identify and visualize endplates 15,16
• Document nAChR cluster formation after myoblast fusion.17
• Label proteins fused to the BBS expression tag (a 13–amino acid
sequence excerpted from the nAChR) in situ 18,19
• Monitor nAChR-mediated responses in neuromuscular damage
and degeneration models 20–22
Biotinylated α-Bungarotoxin
Nicotinic AChRs can also be labeled with biotinylated
α-bungarotoxin (B1196), which is then localized using fluorophore- or
enzyme-labeled avidin, streptavidin or NeutrAvidin biotin-binding
protein conjugates, or NANOGOLD and Alexa Fluor® FluoroNanogold
streptavidin 14,23–25 (Section 7.6, Table 7.9). Based on the intracellular
dissociation of biotinylated α-bungarotoxin and streptavidin, researchers were able to distinguish new, preexisting and recycled pools
of nAChR at the synapses of live mice by sequentially labeling with
biotinylated α-bungarotoxin and fluorescent streptavidin conjugates.26
Complexation of biotinylated α-bungarotoxin with Qdot® nanocrystal–streptavidin conjugates (Section 6.6) enables single-molecule detection of nAChR.1,2 The nanocrystal labeling methodology allows
detection and tracking of diffuse, nonclustered nAChRs, whereas dyelabeled α-bungarotoxin conjugates primarily detect nAChR clusters.1
Table 16.4 Labeled and unlabeled α-bungarotoxins.
Cat. No.
Label
Ex/Em (nm)
Notes
Size
F1176
Fluorescein
494/518
Original green-fluorescent conjugate
500 µg
B13422
Alexa Fluor® 488
495/519
Brightest and most photostable green-fluorescent conjugate
500 µg
T1175
Tetramethylrhodamine
553/577
An extensively used red-orange–fluorescent conjugate
500 µg
B35451
Alexa Fluor® 555
555/565
Bright and photostable red-orange–fluorescent conjugate
500 µg
B13423
Alexa Fluor® 594
590/617
Excellent dye to combine with green-fluorescent probes
500 µg
B35450
Alexa Fluor® 647
650/668
Excellent dye to combine with green- and orange-fluorescent probes
500 µg
B35452
Alexa Fluor® 680
679/702
Excellent dye to combine with green-, orange-, and red-fluorescent probes
500 µg
B1196
Biotin-XX
NA
Visualized with labeled avidins and streptavidins (Table 7.9)
500 µg
B1601
Unlabeled
NA
Useful as a control, as well as for radioiodination and for preparation of new conjugates
1 mg
NA = Not applicable.
The
MolecularProbes®
Probes Handbook:
Handbook: A
Probesand
andLabeling
LabelingTechnologies
Technologies
The
Molecular
A Guide
Guide to
to Fluorescent
Fluorescent Probes
™
760
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 16 — Probes for Endocytosis, Receptors and Ion Channels
Adenylate cyclase–linked
G-protein–coupled receptors
Phospholipase C–linked
G-protein–coupled receptors
RAC/Gi
5-HT1A
5-HT1B
5-HT1D
αt-Adrenergic
Muscarinic
D2 Dopaminergic
A1 Adenosine
Opioid
GABAB
Gs
Gi
+
AC
–
PTX
ATP cAMP+Pi
( )
Cell proliferation.
( )
( )
Gs
5-HT1A (transfected cells)
5-HT1C (transfected cells)
5-HT2 (transfected cells)
α1-Adrenergic
Muscarinic
Metabotropic glutamate
RPLC
RION
RPLC
RAC
PKA
RION
5-HT1A (K+)
5-HT1C (Cl–)
5-HT2 (Cl–)
β-Adrenergic (Ca2+, Na+)
α2-Adrenergic (Ca2+, K+)
Muscarinic (K+, Ca2+)
D2 Dopaminergic (Ca2+, K+)
GABAB (K+)
Gq
Gs
Gi
PIP2 DAG +IP3
PKC
Ligand-gated ion channels
RLG/ION
5-HT3 (Na+, K+)
Nicotinic (Na+)
GABAA (Cl–)
Ionotropic glutamate (Ca2+, Na+)
Ion
channels
RLG/ION
K+
Ca2+
Na+
Cl–
K+
Ca2+
Na+
Cl–
Go
PLC
( )
5-HT1A
5-HT4
β-Adrenergic
D4 Dopaminergic
A2 Adenosine
VIP
Ion channel–linked
G-protein–coupled receptors
( )
RAC/Gs
Section 16.2 Probes for Neurotransmitter Receptors
PTX
Ca2+
Gene expression (cAMP response elements), protein phosphorylation,
changes in process outgrowth, secretion of growth factors from glia.
Calcium can influence cell
proliferation, neurite elongation,
gene expression and cell viability.
Cell proliferation.
Neurite elongation.
Gi
Figure 16.2.1 Neurotransmitter receptors linked to second messengers mediating growth responses in neuronal and nonneuronal cells. Abbreviations: RAC/Gs = Receptors coupled to
G-proteins that stimulate adenylate cyclase (AC) activity, leading to cAMP formation and enhanced activity of protein kinase A (PKA). RAC/Gi = Receptors coupled to pertussis toxin (PTX)–sensitive G-proteins that inhibit adenylate cyclase activity. RPLC = Receptors promoting the hydrolysis of phosphatidylinositol 4,5-diphosphate (PIP2) to inositol 1,4,5-triphosphate (IP3), which increases intracellular Ca2+, and diacylglycerol (DAG), which activates protein kinase C (PKC). RION = Receptors indirectly promoting ion fluxes due to coupling to various G-proteins. RLG/ION = Receptors
that promote ion fluxes directly because they are structurally linked to ion channels (members of the superfamily of ligand-gated ion channel receptors). Stimulation of proliferation is most
often associated with activation of G-proteins negatively coupled to adenylate cyclase (Gi), or positively coupled to phospholipase C (Gq) or to pertussis toxin–sensitive pathways (Go, Gi). In contrast, activation of neurotransmitter receptors positively coupled to cAMP usually inhibits cell proliferation and causes changes in cell shape indicative of differentiation. Reprinted and modified
with permission from J.M. Lauder and Trends Neurosci (1993) 16:233.
( )
( )
In addition, the biotinylated toxin can be employed for affinity isolation of the nAChR using a
streptavidin or CaptAvidin™ agarose (S951, C21386; Section 7.6) column.27,28
Unlabeled α-Bungarotoxin
In addition to the fluorescent and biotinylated derivatives, we have unlabeled α-bungarotoxin
(B1601), which has been shown to be useful for radioiodination.9,29 Unlabeled α-bungarotoxin has
also been employed for ELISA testing of nAChR binding,30 as well as for investigating the function
of the α-bungarotoxin–binding component (α-BgtBC) in vertebrate neurons.31
Amplex® Red Acetylcholine/Acetylcholinesterase Assay Kit
The action of acetylcholine (ACh) at neuromuscular junctions is regulated by acetylcholinesterase (AChE), the enzyme that hydrolyzes ACh to choline and acetate. The Amplex® Red
Acetylcholine/Acetylcholinesterase Assay Kit (A12217) provides an ultrasensitive method for
continuously monitoring AChE activity and for detecting ACh in a fluorescence microplate
reader or fluorometer. Other potential uses for this kit include screening for AChE inhibitors and measuring the release of ACh from synaptosomes. The Amplex® Red Acetylcholine/
Acetylcholinesterase Assay Kit can also be used for the ultrasensitive, specific assay of free
choline, classified as an essential nutrient in foods.32
Figure 16.2.2 Pseudocolored photomicrograph of the
synaptic region of fluorescently labeled living muscle fibers
from the lumbricalis muscle of the adult frog Rana pipiens.
Six hours after isolation of the muscle fibers, acetylcholine
receptors were stained with red-fluorescent tetramethylrhodamine α-bungarotoxin (T1175) and myonuclei were
stained with the green-fluorescent SYTO® 13 live-cell nucleic
acid stain (S7575). Photo contributed by Christian Brösamle,
Brain Research Institute, University of Zurich, and Damien
Kuffler, Institute of Neurobiology, University of Puerto Rico.
™
The
Probes
Handbook:
A Guide
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Probes
and
Labeling
Technologies
TheMolecular
Molecular
Probes®
Handbook:
A Guide
to Fluorescent
Probes
and
Labeling
Technologies
IMPORTANT
NOTICE:described
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or moreUse
Limited
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on on
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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.
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761
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
Figure 16.2.3 Absorption and fluorescence emission spectra of resorufin in pH 9.0 buffer.
In this assay, AChE activity is monitored indirectly using the Amplex® Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), a highly sensitive and stable fluorogenic probe for H 2O2 that
is also useful in assaying other enzymes and analytes (Section 10.5). First, AChE converts the
acetylcholine substrate to choline. Choline is in turn oxidized by choline oxidase to betaine and
H2O2, the latter of which, in the presence of horseradish peroxidase, reacts with the Amplex® Red
reagent to generate the red-fluorescent product resorufin (R363, Section 10.1) with excitation/
emission maxima of ~570/585 nm (Figure 16.2.3). Experiments with purified AChE from electric
eel indicate that the Amplex® Red Acetylcholine/Acetylcholinesterase Assay Kit can detect AChE
levels as low as 0.002 U/mL using a reaction time of only 1 hour (Figure 16.2.4). In our laboratories, we have been able to detect acetylcholinesterase activity from a tissue sample with total
protein content as low as 200 ng/mL or 20 ng/well in a microplate assay.33 By providing an excess
of AChE in the assay, the kit can also be used to detect acetylcholine levels as low as 0.3 µM, with
a detection range between 0.3 µM and ~100 µM acetylcholine (Figure 16.2.5).
The Amplex® Red Acetylcholine/Acetylcholinesterase Assay Kit contains:
•
•
•
•
•
•
•
•
•
Figure 16.2.4 Detection of electric eel acetylcholinesterase activity using the Amplex® Red Acetylcholine/
Acetylcholinesterase Assay Kit (A12217). Each reaction
contained 50 µM acetylcholine, 200 µM Amplex® Red reagent, 1 U/mL HRP, 0.1 U/mL choline oxidase and the indicated amount of acetylcholinesterase in 1X reaction buffer.
Reactions were incubated at room temperature. After 15
and 60 minutes, fluorescence was measured in a fluorescence microplate reader using excitation at 560 ± 10 nm
and fluorescence detection at 590 ± 10 nm. The inset shows
the sensitivity of the 15 min (h) and 60 min (d) assays at
low levels of acetylcholinesterase activity (0–13 mU/mL).
Section 16.2 Probes for Neurotransmitter Receptors
Amplex® Red reagent
Dimethylsulfoxide (DMSO)
Horseradish peroxidase (HRP)
H2O2 for use as a positive control
Concentrated reaction buffer
Choline oxidase from Alcaligenes sp.
Acetylcholine (ACh)
Acetylcholinesterase (AChE) from electric eel
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.
BODIPY® FL Prazosin for α1-Adrenergic Receptors
Prazosin is a high-affinity antagonist for the α1-adrenergic receptor. The green-fluorescent
BODIPY® FL prazosin (B7433, Figure 16.2.6) can be used to localize the α1-adrenergic receptor
on cultured cortical neurons 34 and in vascular smooth muscle cells from α1-adrenergic receptor–knockout mice.35 BODIPY® FL prazosin has also been successfully employed in multidrug
resistance (MDR) transporter activity assays.36,37
3000
Fluorescence
BODIPY® TMR-X Muscimol for GABAA Receptors
60 min
2500
2000
Muscimol is a powerful agonist of the GABA A receptor and has been widely used to reversibly inactivate localized groups of neurons.38,39 Using red-fluorescent BODIPY® TMR-X muscimol (M23400, Figure 16.2.7), researchers can correlate the distribution of muscimol with its
pharmacological effects 40 and detect the presence of GABA A receptors on cell surfaces.41
1200
1500
60 min
15 min
800
1000
400
15 min
500
0
0
0
20
40
0
60
1
2
80
�
100
�
120
Acetylcholine (µM)
Figure 16.2.5 Detection of acetylcholine using the
Amplex® Red Acetylcholine/Acetylcholinesterase Assay Kit
(A12217). Each reaction contained 200 µM Amplex® Red reagent, 1 U/mL HRP, 0.1 U/mL choline oxidase, 0.5 U/mL acetylcholinesterase and the indicated amount of acetylcholine
in 1X reaction buffer. Reactions were incubated at room
temperature. After 15 and 60 minutes, fluorescence was
measured with a fluorescence microplate reader using excitation at 560 ± 10 nm and fluorescence detection at 590 ±
10 nm. The inset shows the sensitivity of the 15 min (h) and
60 min (d) assays at low levels of acetylcholine (0–3 µM).
Fluorescent Angiotensin II for AT1 and AT2 Receptors
Angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) stimulates smooth muscle contraction
and plays an important role in blood pressure control and in water and salt homeostasis. These
effects are exerted via two G-protein–coupled receptor subtypes, referred to as AT1 and AT2.
Our N-terminal–labeled fluorescein and Alexa Fluor® 488 analogs of angiotensin II (A13438,
A13439) are useful tools for imaging the distribution of these receptors,42,43 as well as for flow
cytometric analysis of angiotensin II endocytosis.44 These fluorescent peptides have been characterized for purity by HPLC and mass spectrometry and generally display selectivity for AT1
over AT2 binding.42
The
MolecularProbes®
Probes Handbook:
Handbook: AAGuide
Probesand
andLabeling
LabelingTechnologies
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The
Molecular
Guide to
to Fluorescent
Fluorescent Probes
™
762
IMPORTANT
NOTICE:
The products
described
in this manual
coveredare
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Use Label
License(s).
Please
refer to thePlease
Appendix
onto
IMPORTANT
NOTICE
: The products
described
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by one
or more
Limited
Use Label
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refer
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the Appendix on
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Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
Section 16.2 Probes for Neurotransmitter Receptors
Naloxone Fluorescein for µ-Opioid Receptors
The µ-opioid receptor plays a critical role in analgesia. Among the antagonists that have
been used to define and characterize these receptors are naloxone, a drug used to counteract
the effects of opioid overdose, and naltrexone, a drug used in the treatment of opioid addiction. Naloxone fluorescein (N1384, Figure 16.2.8) has been reported to bind to the µ-opioid
binding site with high affinity,45–47 permitting receptor visualization in transfected Chinese
hamster ovary (CHO) cells.48 Flow cytometry analysis of the binding of naloxone fluorescein to NMDA and µ-opioid receptors (which was displaced by NMDA and met-enkephalin,
respectively) has been used to deduce the effects of operant conditioning on visual cortex
receptor pattern.49
Figure 16.2.6 BODIPY® FL prazosin (B7433).
Probes for Amino Acid Neurotransmitter Receptors
Caged Amino Acid Neurotransmitters
When illuminated with UV light or by multiphoton excitation, caged amino acid neurotransmitters are converted into biologically active amino acids that rapidly initiate neurotransmitter
action.50,51 Thus, these caged probes provide a means of controlling the release—both spatially
and temporally—of agonists for kinetic studies of receptor binding or channel opening.
The different caging groups confer special properties on these photoactivatable probes
(Table 5.2). We synthesize two caged versions of L-glutamic acid 52–60 (C7122, G7055), as well as
caged carbachol 61,62 (N-(CNB-caged) carbachol, C13654) and caged γ-aminobutyric acid 56,63–66
(O-(CNB-caged) GABA, A7110), all of which are biologically inactive before photolysis.67O(CNB-caged) GABA (A7110) and γ-(CNB-caged) L-glutamic acid (G7055), which exhibit fast uncaging rates and high photolysis quantum yields, have been used to investigate the activation kinetics of GABA receptors 66 and glutamate receptors, 55 respectively. N-(CNB-caged) L-glutamic
acid (C7122) does not hydrolyze in aqueous solution because it is caged on the amino group, thus
enabling researchers to use very high concentrations without risk of light-independent glutamic
acid production.55,57
Figure 16.2.7 Muscimol,
(M23400).
BODIPY®
TMR-X
conjugate
Anti–NMDA Receptor Antibodies
N-methyl-D-aspartate (NMDA) receptors constitute cation channels of the central nervous
system that are gated by the excitatory neurotransmitter L-glutamate.68,69 We offer affinity-purified rabbit polyclonal antibodies to NMDA receptor subunits 2A, 2B and 2C (A6473, A6474,
A6475). The anti–NMDA receptor subunit 2A and 2B antibodies were generated against fusion
proteins containing amino acid residues 1253–1391 of subunit 2A and 984–1104 of subunit 2B,
respectively. These two antibodies are active against mouse, rat and human forms of the antigens
and are specific for the subunit against which they were generated. In contrast, the anti–NMDA
receptor subunit 2C antibody was generated against amino acid residues 25–130 of subunit 2C
and recognizes the 140,000-dalton subunit 2C, as well as the 180,000-dalton subunit 2A and
subunit 2B from mouse, rat and human. These three affinity-purified antibodies are suitable for
immunohistochemistry 70 (Figure 16.2.9), western blots, enzyme-linked immunosorbent assays
(ELISAs) and immunoprecipitations.
Figure 16.2.8 Naloxone fluorescein (N1384).
Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit
The Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit (A12221) provides an ultrasensitive method for continuously detecting glutamic acid 71 or for monitoring glutamate
oxidase activity in a fluorescence microplate reader or a fluorometer.72 In this assay, L-glutamic
acid is oxidized by glutamate oxidase to produce α-ketoglutarate, NH3 and H 2O2 . L-Alanine
and L-glutamate–pyruvate transaminase are also included in the reaction. Thus, the L-glutamic
acid is regenerated by transamination of α-ketoglutarate, resulting in multiple cycles of the
initial reaction and a significant amplification of the H 2O2 produced. Hydrogen peroxide reacts
with the Amplex® Red reagent in a 1:1 stoichiometry in a reaction catalyzed by horseradish
peroxidase (HRP) to generate the highly fluorescent product resorufin 73,74 (R363, Section 10.1).
Figure 16.2.9 Rat brain cryosections labeled with antiNMDA receptor, subunit 2A (rat), rabbit IgG fraction (A6473)
and detected using Alexa Fluor® 488 goat anti–rabbit IgG
antibody (A11008). The tissue was also labeled with Alexa
Fluor® 594 anti–glial fibrillary acidic protein antibody
(A21295) and counterstained with TOTO®-3 iodide (T3604),
which was pseudocolored light blue in this image.
™
The
Probes
Handbook:
A Guide
to Fluorescent
Probes
andand
Labeling
Technologies
TheMolecular
Molecular
Probes®
Handbook:
A Guide
to Fluorescent
Probes
Labeling
Technologies
IMPORTANT
NOTICE:described
The products
described
thiscovered
manual are
covered
by one
or moreUse
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Label License(s).
the Appendix
IMPORTANT NOTICE
: The products
in this
manualinare
by one
or more
Limited
LabelUse
License(s).
PleasePlease
refer refer
to thetoAppendix
on on
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763
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
3,500
Fluorescence
3,000
2,500
2,000
200
1,500
150
100
1,000
50
500
0
0
0.025
0.05
0.075
0.1
0
0
5
10
15
20
Glutamic acid (µM)
Figure 16.2.10 Detection of L-glutamic acid using the
Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit
(A12221). Each reaction contained 50 µM Amplex® Red
reagent, 0.125 U/mL HRP, 0.04 U/mL L-glutamate oxidase,
0.25 U/mL L-glutamate–pyruvate transaminase, 100 µM
L-alanine and the indicated amount of L-glutamic acid in
1X reaction buffer. Reactions were incubated at 37°C. After
30 minutes, fluorescence was measured in a fluorescence
microplate reader using excitation at 530 ± 12.5 nm and
fluorescence detection at 590 ± 17.5 nm.
Because resorufin has absorption/emission maxima of ~571/585 nm (Figure 16.2.3), there is
little interference from autofluorescence in most biological samples.
If the concentration of L-glutamic acid is limiting in this assay, then the fluorescence increase
is proportional to the initial L-glutamic acid concentration. The Amplex® Red Glutamic Acid/
Glutamate Oxidase Assay Kit allows detection of as little as 10 nM L-glutamic acid in purified
systems using a 30-minute reaction time (Figure 16.2.10). If the reaction is modified to include
an excess of L-glutamic acid, then this kit can be used to continuously monitor glutamate oxidase
activity. For example, purified L-glutamate oxidase from Streptomyces can be detected at levels
as low as 40 µU/mL (Figure 16.2.11). The Amplex® Red reagent has been used to quantitate the
activity of glutamate-producing enzymes in a high-throughput assay for drug discovery.71 The
Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit contains:
•
•
•
•
•
•
•
•
•
•
Amplex® Red reagent
Dimethylsulfoxide (DMSO)
Horseradish peroxidase (HRP)
H 2 O2
Concentrated reaction buffer
L-Glutamate oxidase from Streptomyces sp.
L-Glutamate–pyruvate transaminase from pig heart
L-Glutamic acid
L-Alanine
Detailed protocols
Each kit provides sufficient reagents for approximately 200 assays using a fluorescence microplate reader and a reaction volume of 100 µL per assay.
6,000
5,000
Fluorescence
Section 16.2 Probes for Neurotransmitter Receptors
4,000
Probes for Other Receptors
600
3,000
500
400
2,000
The Molecular Probes® Handbook discusses a diverse array of receptor probes, including
fluorescent derivatives of:
300
200
100
1,000
0
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
0
0
25
50
75
100
125
Glutamate oxidase (mU/mL)
Figure 16.2.11 Detection of L-glutamate oxidase using
the Amplex® Red Glutamic Acid/Glutamate Oxidase Assay
Kit (A12221). Each reaction contained 50 µM Amplex® Red
reagent, 0.125 U/mL HRP, 0.25 U/mL L-glutamate–pyruvate
transaminase, 20 µM L-glutamic acid, 100 µM L-alanine
and the indicated amount of Streptomyces L-glutamate
oxidase in 1X reaction buffer. Reactions were incubated
at 37°C. After 60 minutes, fluorescence was measured in a
fluorescence microplate reader using excitation at 530 ±
12.5 nm and fluorescence detection at 590 ± 17.5 nm. The
inset represents data from a separate experiment for lower
L-glutamate oxidase concentrations and incubation time of
60 minutes (0–1.25 mU/mL).
•
•
•
•
•
•
Low-density lipoprotein (LDL)
Lipopolysaccharides
Epidermal growth factor (EGF)
Transferrin
Fibrinogen
Gelatin and collagen
•
•
•
•
•
•
Ovalbumin and bovine serum albumin
Casein
Histone H1
Subunit B of cholera toxin
Chemotactic peptide
Insulin
These ligands are all transported into the cell by receptor-mediated endocytosis. Additional
information about these probes, as well as membrane and fluid-phase markers, can be found in
Section 16.1.
REFERENCES
1. BMC Neurosci (2009) 10:80; 2. Nano Lett (2008) 8:780; 3. Proc Natl Acad Sci U S A (2007) 104:13666; 4. Am J Physiol
Cell Physiol (2006) 290:C728; 5. J Cell Biol (2005) 170:619; 6. J Immunol Methods (2004) 289:169; 7. J Histochem
Cytochem (2006) 54:817; 8. Biochemistry (1990) 29:11009; 9. Meth Neurosci (1992) 8:67; 10. J Cell Biol (1998) 141:1613;
11. Proc Natl Acad Sci U S A (1976) 73:4594; 12. J Physiol (1974) 237:385; 13. J Cell Biol (1993) 120:197; 14. J Cell Biol
(1993) 120:185; 15. J Neurosci (1995) 15:520; 16. J Cell Biol (1994) 124:139; 17. Biophys J (2006) 90:2192; 18. Proc Natl
Acad Sci U S A (2004) 101:17114; 19. J Biol Chem (2008) 283:15160; 20. J Orthop Res (2009) 27:114; 21. J Neurosci
(2006) 26:6873; 22. J Clin Invest (2004) 113:265; 23. J Cell Biol (1995) 131:441; 24. J Biol Chem (1993) 268:25108;
25. Proc Natl Acad Sci U S A (1980) 77:4823; 26. Clin Chim Acta (2007) 379:119; 27. J Neurosci (2008) 28:11468;
28. Mol Brain (2008) 1:18; 29. Biochemistry (1979) 18:1875; 30. Toxicon (1991) 29:503; 31. Neuron (1992) 8:353;
32. Science (1998) 281:794; 33. Proc SPIE-Int Soc Opt Eng (2000) 3926:166; 34. Brain Res Dev Brain Res (1997) 102:35;
35. Br J Pharmacol (2009) 158:209; 36. J Pharmacol Exp Ther (2009) 331:1118; 37. Br J Pharmacol (2004) 143:899; 38. J
Neurosci Res (1992) 31:166; 39. Neural Plast (2000) 7:19; 40. J Neurosci Methods (2008) 171:30; 41. Proc Natl Acad Sci
U S A (2007) 104:335; 42. Am J Physiol Renal Physiol (2006) 291:F375; 43. J Neurosci Methods (2005) 143:3; 44. Am
J Physiol Renal Physiol (2005) 288:F420; 45. Pharm Res (1986) 3:56; 46. Pharm Res (1985) 6:266; 47. Life Sci (1983)
33 Suppl 1:423; 48. J Neurosci Methods (2000) 97:123; 49. Biol Chem Hoppe Seyler (1995) 376:483; 50. Nat Methods
TheMolecular
MolecularProbes®
Probes Handbook:
Handbook: AAGuide
and Labeling
LabelingTechnologies
Technologies
The
Guideto
toFluorescent
Fluorescent Probes
Probes and
™
764
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 16 — Probes for Endocytosis, Receptors and Ion Channels
Section 16.2 Probes for Neurotransmitter Receptors
REFERENCES—continued
(2007) 4:619; 51. J Neurosci Methods (2004) 133:153; 52. Nat Neurosci (1998) 1:119;
53. Neuroscience (1998) 86:265; 54. Science (1998) 279:1203; 55. Proc Natl Acad Sci U S
A (1994) 91:8752; 56. J Org Chem (1996) 61:1228; 57. Abstr Soc Neurosci (1995) 21:579,
abstract 238.11; 58. J Neurosci Methods (1994) 54:205; 59. Science (1994) 265:255;
60. Proc Natl Acad Sci U S A (1993) 90:7661; 61. J Neurosci (2003) 23:9024; 62. Proc
Natl Acad Sci U S A (2000) 97:13895; 63. Methods Enzymol (1998) 291:443; 64. Neuron
(1995) 15:755; 65. J Org Chem (1990) 55:1585; 66. J Am Chem Soc (1994) 116:8366;
67. Methods Enzymol (1998) 291:30; 68. Neuron (1994) 12:529; 69. Nature (1991) 354:31;
70. J Neurochem (2000) 75:2040; 71. Anal Biochem (2000) 284:382; 72. Anal Chim Acta
(1999) 402:47; 73. Anal Biochem (1997) 253:162; 74. J Immunol Methods (1997) 202:133.
DATA TABLE 16.2 PROBES FOR NEUROTRANSMITTER RECEPTORS
Cat. No.
MW
Storage
Soluble
Abs
EC
Em
Solvent
Notes
262
4500
none
pH 7
1, 2
A7110
396.28
F,D,LL
H2O
494
78,000
522
pH 9
3
A13438
1404.50
F,D,L
H2O, DMSO
491
78,000
516
pH 7
3
A13439
1586.64
F,D,L
H2O, DMSO
<300
none
4
B1196
~8400
F,D
H2O
<300
see Notes
5
B1601
7984.14
F
H2O
B7433
563.41
F,D,L
DMSO, EtOH
504
77,000
511
MeOH
495
78,000
519
pH 8
4, 6
B13422
9000
F,D,L
H2O
593
92,000
617
pH 7
4, 6
B13423
9000
F,D,L
H2O
649
246,000
668
pH 7
4, 6
B35450
9000
F,D,L
H2O
554
150,000
567
pH 7
4, 6
B35451
9000
F,D,L
H2O
680
180,000
704
pH 7
4, 6
B35452
9000
F,D,L
H2O
266
4800
none
pH 7
1, 2
C7122
326.26
F,D,LL
H2O
264
4200
none
H2O
1, 2
C13654
439.34
F,D,LL
H2O
494
84,000
518
pH 8
4, 6
F1176
9000
F,D,L
H2O
262
5100
none
pH 7
1, 2
G7055
440.29
F,D,LL
H2O, DMSO
M23400
607.46
F,D,L
DMSO
543
60,000
572
MeOH
N1384
790.84
D,L
EtOH, DMF
492
79,000
516
pH 9
553
85,000
577
H2O
4, 6
T1175
9000
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. All photoactivatable probes are sensitive to light. They should be protected from illumination except when photolysis is intended.
2. This compound has weaker visible absorption at >300 nm but no discernible absorption peaks in this region.
3. The value of EC listed for this peptide conjugate is that of the labeling dye in free solution. Use of this value for the conjugate assumes a 1:1 dye:peptide labeling ratio and no change of EC due to
dye–peptide interactions.
4. α-Bungarotoxin, EGF and phallotoxin conjugates have approximately 1 label per peptide.
5. This peptide exhibits intrinsic tryptophan fluorescence (Em ~350 nm) when excited at <300 nm.
6. The value of EC listed for this α-bungarotoxin conjugate is for the labeling dye in free solution. Use of this value for the conjugate assumes a 1:1 dye:peptide labeling ratio and no change of EC
due to dye–peptide interactions.
PRODUCT LIST 16.2 PROBES FOR NEUROTRANSMITTER RECEPTORS
Cat. No.
Product
Quantity
A7110
A12217
A12221
A13439
A13438
A6473
A6474
A6475
B7433
B1601
B13422
B35451
B13423
B35450
B35452
B1196
C13654
C7122
F1176
G7055
M23400
N1384
T1175
γ-aminobutyric acid, α-carboxy-2-nitrobenzyl ester, trifluoroacetic acid salt (O-(CNB-caged) GABA)
Amplex® Red Acetylcholine/Acetylcholinesterase Assay Kit *500 assays*
Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit *200 assays*
angiotensin II, Alexa Fluor® 488 conjugate
angiotensin II, fluorescein conjugate
anti-NMDA receptor, subunit 2A (rat), rabbit IgG fraction *affinity purified*
anti-NMDA receptor, subunit 2B (rat), rabbit IgG fraction *affinity purified*
anti-NMDA receptor, subunit 2C (rat), rabbit IgG fraction *affinity purified*
BODIPY® FL prazosin
α-bungarotoxin *from Bungarus multicinctus*
α-bungarotoxin, Alexa Fluor® 488 conjugate
α-bungarotoxin, Alexa Fluor® 555 conjugate
α-bungarotoxin, Alexa Fluor® 594 conjugate
α-bungarotoxin, Alexa Fluor® 647 conjugate
α-bungarotoxin, Alexa Fluor® 680 conjugate
α-bungarotoxin, biotin-XX conjugate
N-(CNB-caged) carbachol (N-(α-carboxy-2-nitrobenzyl)carbamylcholine, trifluoroacetic acid salt)
N-(CNB-caged) L-glutamic acid (N-(α-carboxy-2-nitrobenzyl)-L-glutamic acid)
fluorescein α-bungarotoxin (α-bungarotoxin, fluorescein conjugate)
L-glutamic acid, γ-(α-carboxy-2-nitrobenzyl) ester, trifluoroacetic acid salt (γ-(CNB-caged) L-glutamic acid)
muscimol, BODIPY® TMR-X conjugate
naloxone fluorescein
tetramethylrhodamine α-bungarotoxin (α-bungarotoxin, tetramethylrhodamine conjugate)
5 mg
1 kit
1 kit
25 µg
25 µg
10 µg
10 µg
10 µg
100 µg
1 mg
500 µg
500 µg
500 µg
500 µg
500 µg
500 µg
5 mg
5 mg
500 µg
5 mg
1 mg
5 mg
500 µg
™
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
765
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
Section 16.3 Probes for Protein Kinases, Protein Phosphatases
and Nucleotide-Binding Proteins
16.3 Probes for Ion Channels and Carriers
This section describes a variety of probes for Ca 2+, Na+, K+ and Cl– ion channels and carriers.
Chapter 19 and Chapter 21 contain our extensive selection of indicators for these physiologically important ions, providing a means of correlating ion channel activation with subsequent
changes in intracellular ion concentration. Ion flux also affects the cell’s membrane potential,
which can be measured with the probes described in Chapter 22.
Probes for Ca2+ Channels and Carriers
Figure 16.3.1 DM-BODIPY® (–)-dihydropyridine (D7443).
In both excitable and nonexcitable cells, intracellular Ca 2+ levels modulate a multitude of
vital cellular processes—including gene expression, cell viability, cell proliferation, cell motility
and cell shape and volume regulation—and thereby play a key role in regulating cell responses
to external activating agents. These dynamic changes in intracellular Ca 2+ levels are regulated
by ligand-gated and G-protein–coupled ion channels in the plasma membrane, as well as by mobilization of Ca 2+ from intracellular stores. One of the best-studied examples of Ca 2+-dependent
signal transduction is the depolarization of excitable cells, such as those of neuronal, cardiac,
skeletal and smooth muscle tissue, which is mediated by inward Ca 2+ and Na+ currents. The
Ca 2+ current is attributed to the movement of ions through N-, L-, P- and T-type Ca 2+ channels, which are defined both pharmacologically and by their biophysical properties, including
conductance and voltage sensitivity. Here we describe several fluorescent ligands for imaging
the spatial distribution and localization of Ca 2+ channels in cells, as well as Premo™ Cameleon
Calcium Sensor, a genetically encoded, protein-based ratiometric sensor for calcium measurements. Our complete selection of Ca 2+ indicators is described in Chapter 19.
Fluorescent Dihydropyridine for L-Type Ca2+ Channels
Figure 16.3.2 BODIPY® FL verapamil, hydrochloride (B7431).
480 nm
440 nm
The L-type Ca 2+ channel is readily blocked by the binding of dihydropyridine to the channel’s pore-forming α1-subunit. To facilitate the study of channel number and distribution in
single cells, we offer fluorescent dihydropyridine derivatives. The high-affinity (–)-enantiomer
of dihydropyridine is available labeled with either the green-fluorescent DM-BODIPY® (D7443,
Figure 16.3.1) or the orange-fluorescent ST-BODIPY® (S7445) fluorophore. Knaus and colleagues
have shown that these BODIPY® dihydropyridines bind to L-type Ca 2+ channels with high affinity and inhibit the Ca 2+ influx in GH3 cells.1–3 For neuronal L-type Ca 2+ channels, the (–)-enantiomers of the DM-BODIPY® dihydropyridine and ST-BODIPY® derivatives each exhibit a
K i of 0.9 nM. Their affinities for skeletal muscle L-type Ca 2+ channels are somewhat lower.
Although DM-BODIPY® dihydropyridine exhibits a more intense fluorescence, the particularly
high degree of stereoselectivity retained by the ST-BODIPY® derivatives has proven useful for
the in vivo visualization of L-type Ca 2+ channels.4 DM-BODIPY® dihydropyridine has proven
effective as a substrate for functional analysis of ABC drug transporters. 5
BODIPY® FL Verapamil
+ 4 Ca2+
FRET
535 nm
440 nm
Figure 16.3.4 Schematic of the Premo™ Cameleon Calcium
Sensor (P36207, P36208) mechanism.
Like dihydropyridine, phenylalkylamines also bind to the α1-subunit of L-type Ca 2+ channels and block Ca 2+ transport. We offer a green-fluorescent BODIPY® FL derivative (B7431,
Figure 16.3.2) of verapamil, a phenylalkylamine known to inhibit P-glycoprotein–mediated
drug efflux.
The 170,000-dalton P-glycoprotein is typically overexpressed in tumor cells that have acquired resistance to a variety of anticancer drugs (Section 15.6). P-glycoprotein is thought to
mediate the ATP-dependent efflux or sequestration of structurally unrelated molecules, including actinomycin D, anthracyclines, colchicine, epipodophyllotoxins and vinblastine. Verapamil
appears to inhibit drug efflux by acting as a substrate of P-glycoprotein, thereby overwhelming
the transporter’s capacity to expel the drugs. BODIPY® FL verapamil also appears to serve as a
substrate for P-glycoprotein. This fluorescent verapamil derivative preferentially accumulates
in the lysosomes of normal, drug-sensitive NIH 3T3 cells but is rapidly transported out of
multidrug-resistant cells.6–9
The
MolecularProbes®
Probes Handbook:
Handbook: AA Guide
Probesand
andLabeling
LabelingTechnologies
Technologies
The
Molecular
Guide to
to Fluorescent
Fluorescent Probes
™
766
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 16 — Probes for Endocytosis, Receptors and Ion Channels
Section 16.3 Probes for Protein Kinases, Protein Phosphatases
and Nucleotide-Binding Proteins
Eosin Derivatives: Inhibitors of the Calcium Pump
Eosin isothiocyanate (E18) is a potent reversible inhibitor of the erythrocyte plasma membrane calcium pump, with a half-maximal inhibitory concentration of <0.2 µM.10 Eosin isothiocyanate also reacts irreversibly at the ATP-binding site of this calcium pump. The succinimidyl ester of carboxyeosin diacetate (C22803), a cell membrane–permeant eosin derivative, also
inhibits the erythrocyte plasma membrane Ca 2+ pump.11,12 Fluorescein isothiocyanate (F143,
Section 1.4) is a weaker inhibitor of the erythrocyte plasma membrane calcium pump.
Fluorescence emission
Ex = 435 nm
Premo™ Cameleon Calcium Sensor
450
500
550
600
Wavelength (nm)
Figure 16.3.5 Fluorescence emission spectra of Premo™
Cameleon Calcium Sensor (P36207, P36208). The dashed
line indicates the spectra in the absence of Ca2+; the solid
line shows the fluorescence resonance energy transfer
(FRET)–based change upon Ca2+ binding.
1.75
YFP/CFP ratio (max–min)
A
1.50
1.25
1.00
0.75
0.50
0.25
0
0.01
0.1
1
10
100
Histamine (µM)
2.25
B
YFP/CFP ratio (max–min)
The Premo™ product line combines genetically encoded ion indicators and environmental
sensors with efficient BacMam delivery (BacMam Gene Delivery and Expression Technology—
Note 11.1) for intracellular measurements in mammalian cells. Premo™ Cameleon Calcium
Sensor (P36207, P36208) is a ratiometric calcium-sensitive fluorescent protein that is delivered by
BacMam baculovirus-mediated transduction to a variety of mammalian cell types. This content
and delivery system provides an effective and robust technique for measuring Ca 2+ mobilization
in transduced cells using microplate assays or fluorescence microscopy (Figure 16.3.3).
The Premo™ Cameleon Calcium Sensor is based on the YC3.60 version of the fluorescent
protein (FP)–based sensor (cameleon) family developed by Tsien, Miyawaki and co-workers,
which is reported to have a Ca 2+ dissociation constant of 240 nM.13,14 The sensor comprises
two fluorescent proteins (Enhanced Cyan Fluorescent Protein or ECFP and Venus variant
of Yellow Fluorescent Protein or YFP), linked by the calmodulin-binding peptide M13 and
calmodulin. Upon binding four calcium ions, calmodulin undergoes a conformational change
by wrapping itself around the M13 peptide, which changes the efficiency of the fluorescence
resonance energy transfer (FRET) between the CFP donor and the YFP acceptor fluorophores
(Figure 16.3.4). Following this conformational change, there is an increase in YFP emission
(525–560 nm) and a simultaneous decrease in CFP emission (460–500 nm) (Figure 16.3.5),
making Cameleon an effective reporter of calcium mobilization. The ratiometric readout of
the Premo™ Cameleon Calcium Sensor—an increase in YFP emission (535 nm, green-yellow
emission) and a decrease in CFP emission (485 nm, blue emission)—reduces assay variations
due to compound or cellular autofluorescence, nonuniform cell plating, differences in expression levels between cells, instability of instrument illumination and changes in illumination
pathlength.
The Premo™ Cameleon Calcium Sensor is designed to readily and accurately detect intracellular calcium flux from different receptors. Standard pharmacological assays for multiple GPCR
agonists and antagonists have been tested. An example of the robustness and reproducibility and
accuracy of the system is demonstrated using the endogenous histamine receptor in conjunction
with histamine, pyrilamine, and thioperamide in HeLa cells (Figure 16.3.6). Expression levels
will be maintained for several days, enabling iterative assays to be run, for instance, when examining agonist/antagonist relationships on the same cells.
2.00
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0
0.001
0.01
0.1
1
10
100
1,000
Antagonist (µM)
Figure 16.3.6 Agonist and antagonist dose response
curves. HeLa cells were plated in a 96-well plate at a density of 15,000 cells/well, transduced with Premo™ Cameleon
Calcium Sensor (P36207, P36208), and incubated overnight
at 37°C. The following day, a histamine dose response was
performed (A). A separate plate was used to evaluate an
antagonist dose response with pyrilamine (j) and thioperamide (m) in the presence of an EC80 concentration of
histamine (B). Pyrilamine is a known H1 receptor antagonist
that couples through Gq proteins and the second messenger Ca2+. Thioperamide is a known H3 receptor antagonist
that couples through Gi proteins and the second messenger cAMP.
Figure 16.3.3 Porcine left atrial appendage progenitor cells were transfected with Premo™ Cameleon calcium sensor (P36207,
P36208); ATP (20 µM final concentration) was applied to the cells the following day and the cells were imaged using a Zeiss
5 Live high-speed confocal system (Carl Zeiss MicroImaging). Excitation was with a 405 nm diode laser (50 mw) operated at
50% power. Emission was collected simultaneously on two linear CCD detectors using a 490 nm dichroic mirror to split the
beam through a 415–480 nm bandpass filter for CFP and a 550 nm longpass filter for YFP. Images were collected at a rate of 10
frames per second (512 x 512 pixels) using a 40x Plan-Neofluar 1.3 NA oil immersion objective lens.
™
The
Probes
Handbook:
A Guide
to Fluorescent
Probes
andand
Labeling
Technologies
TheMolecular
Molecular
Probes®
Handbook:
A Guide
to Fluorescent
Probes
Labeling
Technologies
IMPORTANT
NOTICE:described
The products
described
thiscovered
manual are
covered
by one
or more
Limited
Label License(s).
the Appendix
IMPORTANT NOTICE
: The products
in this
manualinare
by one
or more
Limited
Use
LabelUse
License(s).
PleasePlease
refer 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.
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thermofisher.com/probes
767
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
Section 16.3 Probes for Protein Kinases, Protein Phosphatases
and Nucleotide-Binding Proteins
Probes for Na+ Channels and Carriers
Amiloride Analogs: Probes for the Na+ Channel and the Na+/H+ Antiporter
Figure 16.3.7 5-(N-ethyl-N-isopropyl)amiloride, hydrochloride (E3111).
Amiloride is a compound known to inhibit the Na+/H+ antiporter of vertebrate cells by acting competitively at the Na+-binding site.15 The antiporter extrudes protons from cells using the
inward Na+ gradient as a driving force, resulting in intracellular alkalinization. In 1967, Cragoe
and co-workers reported the synthesis of amiloride and several amiloride analogs, which are pyrazine diuretics that inhibit the Na+ channel in urinary epithelia.16 Since then, more than 1000 different amiloride analogs have been synthesized and many of these tested for their specificity and
potency in inhibiting the Na+ channel, Na+/H+ antiporter and Na+/Ca 2+ exchanger.17 Unmodified
amiloride inhibits the Na+ channel with an IC50 of less than 1 µM. Additionally, amiloride is an
important tool for studying the Na+/H+ antiporter. Structure–activity relationships have demonstrated that amiloride analogs with hydrophobic groups in the drug are the most potent and
specific inhibitors for the Na+/H+ antiporter.17–22 For example, 5-(N-ethyl-N-isopropyl)amiloride
(EIPA, E3111; Figure 16.3.7) is 200-fold more potent than amiloride for inhibiting this antiporter.
Ouabain Probes for Na+/K+-ATPase
Ouabain is a member of a class of glycosylated steroids collectively known as cardiac glycosides due to their therapeutic efficacy in the treatment of congestive heart failure. Ouabain
achieves this effect by binding to the catalytic α-subunit of Na+/K+-ATPase and inhibiting its
transport of Na+ across the plasma membrane. 9-Anthroyl ouabain (A1322) is useful for localizing Na+/K+-ATPase and for studying its membrane orientation, mobility and dynamics.23
Anthroyl ouabain has also been employed to investigate Na+/K+-ATPase’s active site, inhibition
and conformational changes,24–29 as well as to investigate the kinetics of cardiac glycoside binding.30–35 BODIPY® FL ouabain (B23461, Figure 16.3.8) has been used in combination with Alexa
Fluor® 555 cholera toxin B (C22843, Section 16.1) for visualizing Na+/K+-ATPase and ganglioside
GM1 domain localization in lymphocyte plasma membranes.36
Figure 16.3.8 BODIPY® FL ouabain (B23461).
Figure 16.3.9 ER-Tracker™ Green (BODIPY® FL glibenclamide, E34251).
Using BacMam Technology to Deliver and Express Sodium Channel cDNA
Sodium channel cDNAs that have been engineered into a baculovirus gene delivery/expression system using BacMam technology (BacMam Gene Delivery and Expression Technology—
Note 11.1) are also available, including the Nav1.2 cDNA (B10341) and the Nav1.5 cDNA (B10335).
The BacMam system uses a modified insect cell baculovirus as a vehicle to efficiently deliver
and express genes in mammalian cells with minimum effort and toxicity. The use of BacMam
delivery in mammalian cells is relatively new, but well described, and has been used extensively
in a drug discovery setting.37 Furthermore, constitutively expressed ion channels and other cell
surface proteins have been shown to contribute to cell toxicity in some systems, and may be
subject to clonal drift and other inconsistencies that hamper successful experimentation and
screening. Thus, transient expression systems such as the BacMam gene delivery and expression
system are increasingly methods of choice to decrease variability of expression in such assays.
U2OS cells (ATCC number HTB-96) have been shown to demonstrate highly efficient expression of BacMam delivered targets in a null background ideal for screening in a heterologous
expression system. The U2OS cell line is recommended for use if your particular cell line does not
efficiently express the BacMam targets. Examples of other cell lines that are efficiently transduced
by BacMam technology include HEK 293, HepG2, BHK, Cos-7 and Saos-2.
Probes for K+ Channels and Carriers
Glibenclamide Probes for the ATP-Dependent K+ Channel
Glibenclamide blocks the ATP-dependent K+ channel, thereby eliciting insulin secretion.38
We have prepared the green-fluorescent BODIPY® FL glibenclamide (BODIPY® FL glyburide,
E34251; Figure 16.3.9) and red-fluorescent BODIPY® TR glibenclamide (BODIPY® TR glyburide,
E34250) as probes for the ATP-dependent K+ channel. BODIPY® TR glibenclamide has been used
to detect sulfonylurea receptors associated with ATP-dependent K+ channels in bovine monocytes
and in β-cells 39,40 and to label a novel mitochondrial ATP-sensitive potassium channel in brain.41
The sulfonylurea receptors of ATP-dependent K+ channels are prominent on the endoplasmic reticulum (ER). Therefore, because these probes are also effective live-cell stains for ER,
The
MolecularProbes®
Probes Handbook:
Handbook: A
Probesand
andLabeling
LabelingTechnologies
Technologies
The
Molecular
A Guide
Guide to
to Fluorescent
Fluorescent Probes
™
768
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 16 — Probes for Endocytosis, Receptors and Ion Channels
Section 16.3 Probes for Protein Kinases, Protein Phosphatases
and Nucleotide-Binding Proteins
BODIPY® FL glibenclamide and BODIPY® TR glibenclamide are also
referred to as ER-Tracker™ Green and ER-Tracker™ Red, respectively; see
Section 12.4 for a description of this application. Variable expression
of sulfonylurea receptors in some specialized cell types may result in
non-ER labeling with these probes.
co-transport processes that accommodate the transport of thallium into
cells.44 Furthermore, resting potassium channels and inward rectifier potassium channels like Kir2.1 can be assayed by adding stimulus buffer
with thallium alone, without any depolarization to measure the signal.
The FluxOR™ reagent, a thallium indicator dye, is loaded into cells
as a membrane-permeable AM ester. Loading is assisted by the proprietary PowerLoad™ concentrate, an optimized formulation of nonionic
Pluronic® surfactant polyols that act to disperse and stabilize AM ester
dyes for optimal loading in aqueous solution. This PowerLoad™ concentrate is also available separately (P10020) to aid the solubilization of
water-insoluble dyes and other materials in physiological media.
Once inside the cell, the nonfluorescent AM ester of the FluxOR™
dye is cleaved by endogenous esterases into a weakly fluorescent (basal
fluorescence), thallium-sensitive indicator. The thallium-sensitive form
is retained in the cytosol, and its extrusion is inhibited by water-soluble
probenecid (P36400, Section 19.8), which blocks organic anion pumps.
For most applications, cells are loaded with the dye at room temperature. For best results, the dye-loading buffer is then replaced with fresh,
dye-free assay buffer (composed of physiological HBSS containing probenecid), and cells are ready for the high-throughput screening assay.
Each FluxOR™ Potassium Ion Channel Assay Kit contains:
FluxOR™ Potassium Ion Channel Assay
The FluxOR™ Potassium Ion Channel Assay Kits (F10016, F10017)
provide a fluorescence-based assay for high-throughput screening of
potassium ion channel and transporter activities.42,43 The FluxOR™
Potassium Ion Channel Assay Kits take advantage of the well-described
permeability of potassium channels to thallium (Tl+) ions. When thallium
is present in the extracellular solution containing a stimulus to open potassium channels, channel activity is detected with a cell-permeant thallium indicator dye that reports large increases in fluorescence emission
at 525 nm as thallium flows down its concentration gradient and into the
cells (Figure 16.3.10). In this way, the fluorescence reported in the FluxOR™
system becomes a surrogate indicator of activity for any ion channel or
transporter that is permeable to thallium, including the human ethera-go-go–related (hERG) channel, one of the human cardiac potassium
channels. The FluxOR™ potassium ion channel assay has been validated
for homogeneous high-throughput profiling of hERG channel inhibition
using BacMam-mediated transient expression of hERG.42 The FluxOR™
Potassium Ion Channel Assay Kits can also be used to study potassium
Resting
Stimulated
+
+
Tl
Dye
+
+
Tl
Tl
+
Tl
Thallium
+
Tl
•
•
•
•
•
•
•
•
•
+
Tl
Tl
+
Tl
+
Tl
Ion channel
Tl
Closed
Tl
+
+
FluxOR™ reagent
FluxOR™ assay buffer
PowerLoad™ concentrate
Probenecid
FluxOR™ chloride-free buffer
Potassium sulfate (K 2SO4) concentrate
Thallium sulfate (Tl2SO4) concentrate
Dimethylsulfoxide (DMSO)
Detailed protocols
Ion channel
+
Figure 16.3.10 Thallium redistribution in the FluxOR™ assay. Basal fluorescence from cells
loaded with FluxOR™ reagent (provided in the FluxOR™ Potassium Ion Channel Assay Kits;
F10016, F10017) is low when potassium channels remain unstimulated, as shown in the left
panel. When thallium is added to the assay with the stimulus, the thallium flows down its
concentration gradient into the cells, activating the dye as shown in the right panel.
The FluxOR™ Kits provide a concentrated thallium solution along
with sufficient dye and buffers to perform ~4000 (F10016) or ~40,000
(F10017) assays in a 384-well microplate format. These kits allow maximum target flexibility and ease of operation in a homogeneous format.
The FluxOR™ potassium ion channel assay has been demonstrated
for use with CHO and HEK 293 cells stably expressing hERG, as well
as U2OS cells transiently transduced with BacMam hERG reagent 42
(B10019, B10033) (Figure 16.3.11). More information is available at
www.invitrogen.com/handbook/fluxorpotassium.
A
B 2.5
C 2.0
2.3
1.8
Tl
+
Tl
+
+
Tl
Tl
Tl
Intracellular
Extracellular
39
34
U-2 OS
BacMam hERG
∆ F/F
1.9
24
1.7
1.5
19
Cisapride block or
BacMam negative control
1.3
14
9
+
Intracellular
2.1
U-2 OS
BacMam hERG
29
∆ F/F
103 RFU
(Relative fluorescence units)
Extracellular
Cisapride block or
BacMam negative control
1.1
0
20
40
60
Time (sec)
80
100
120
0.9
D
IC50 = 73 nM
1.6
1.6
1.4
1.4
1.2
1.2
1.0
20
40
60
Time (sec)
80
100
120
0.8
0.6
0.6
0.2
IC50 = 79 nM
Frozen
1.0
0.8
0.4
0
2.0
1.8
Fresh
∆ F/F
+
Tl
Open
+
Tl
0.4
10-1
100
101
102
103
[Cisapride] (nM)
104
105
0.2
10-1
100
101
102
103
104
105
[Cisapride] (nM)
Figure 16.3.11 FluxOR™ potassium ion channel assays (F10016, F10017) performed on fresh and frozen U2OS cells transduced with the BacMam hERG reagent (B10019, B10033). A) Raw data obtained in the FluxOR™ assay determination of thallium flux in U2OS cells transduced with BacMam-hERG and kept frozen until the day of use. The arrow indicates the addition of the thallium/potassium stimulus, and upper and lower traces indicate data taken from the minimum and maximum doses of cisapride used in the determination of the dose-response curves. B) Raw pre-stimulus
peak and baseline values were boxcar averaged and normalized, and indicate the fold increase in fluorescence over time. C) Data generated in a dose-response determination of cisapride block
on BacMam hERG expressed in U2OS cells freshly prepared from overnight expression after viral transduction. D) Parallel data obtained from cells transduced with BacMam-hERG, stored for 2
weeks in liquid nitrogen, thawed, and plated 4 hours prior to running the assay. Error bars indicate standard deviation, n = 4 per determination.
™
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
769
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
Section 16.3 Probes for Protein Kinases, Protein Phosphatases
and Nucleotide-Binding Proteins
Using BacMam Technology to Deliver and
Express Potassium Channel cDNA
Potassium channel cDNAs that have been engineered into a baculovirus gene delivery/expression system using BacMam technology
(BacMam Gene Delivery and Expression Technology—Note 11.1) are
also available for use with the FluxOR™ Potassium Ion Channel Assay
Kits, including the human ether-a-go-go related gene (hERG) (Figure
16.3.12), several members of the voltage-gated K+ channel (Kv) gene
family and two members of the inwardly rectifying K+ channel (Kir)
gene family:
• BacMam hERG 42 (for 10 microplates, B10019; for 100 microplates,
B10033)
• BacMam Kv1.1 (for 10 microplates, B10331)
• BacMam Kv1.3 (for 10 microplates, B10332)
• BacMam Kv2.1 (for 10 microplates, B10333)
• BacMam Kv7.2 and Kv7.3 (for 10 microplates, B10147)
• BacMam Kir1.1 (for 10 microplates, B10334)
• BacMam Kir2.1 (for 10 microplates, B10146)
The BacMam system uses a modified insect cell baculovirus as a
vehicle to efficiently deliver and express genes in mammalian cells with
minimum effort and toxicity. The use of BacMam delivery in mammalian cells is relatively new, but well described, and has been used
extensively in a drug discovery setting.37 Furthermore, constitutively
expressed ion channels and other cell surface proteins have been shown
to contribute to cell toxicity in some systems, and may be subject to
clonal drift and other inconsistencies that hamper successful experimentation and screening. Thus, transient expression systems such as
BacMam technology are increasingly methods of choice to decrease
variability of expression in such assays.
Promoter
U2OS cells (ATCC number HTB-96) have been shown to demonstrate highly efficient expression of BacMam-delivered targets in a null
background ideal for screening in a heterologous expression system.
The U2OS cell line is recommended for use if your particular cell line
does not efficiently express the BacMam targets. Examples of other cell
lines that are efficiently transduced by BacMam technology include
HEK 293, HepG2, BHK, Cos-7 and Saos-2.
Probes for Anion Transporters
Stilbene Disulfonates: Anion-Transport Inhibitors
We offer three stilbene disulfonates that have been employed to
inhibit (frequently irreversibly) anion transport 45 in a large number of
mammalian cell types:
• DIDS (D337, Figure 16.3.13)
• H2DIDS (D338)
• DNDS (D673)
Our stilbene disulfonate probes, which are 95–99% pure by HPLC,
have significantly higher purity and more defined composition than
those available from other commercial sources. DNDS was among the
inhibitors used to characterize three different anion exchangers in the
membranes of renal brush border cells and to compare these exchangers
with the band-3 anion-transport protein of erythrocyte membranes.46
These stilbene disulfonates can, in some cases, bind specifically
to proteins that are not anion transporters. For example, DIDS and
H2DIDS complex specifically with the CD4 glycoprotein on T-helper
lymphocytes and macrophages, blocking HIV type-1 growth at multiple stages of the virus life cycle.47
human Ether-à-go-go Related Gene
hERG Gene
Ion channel
Membrane
insertion
Assembly
S C �
mRNA
translated
Baculovirus
CH
CH
S��
� C S
��S
����
Endocytotic entry
mRNA
Figure 16.3.13 DIDS (4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid, disodium salt, D337).
DNA
DNA moves
to nucleus
hERG gene
transcribed
Figure 16.3.12 BacMam-hERG gene delivery and expression. This schematic depicts the
mechanism of BacMam-mediated gene delivery into a mammalian cell and expression of
the hERG gene (B10019, B10033). The hERG gene resides within the baculoviral DNA, downstream of a CMV promoter that drives its expression when introduced into a mammalian
target cell. BacMam viral particles are taken up by endocytic pathways into the cell, and the
DNA within them is released for transcription and expression. The translated protein is then
folded for insertion into the membrane, forming functional hERG ion channels. This process
begins within 4–6 hours and in many cell types is completed after an overnight period.
Figure 16.3.14 Bis-(1,3-dibutylbarbituric acid)pentamethine oxonol (DiBAC4(5), B436).
The
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Probes Handbook:
Handbook: AAGuide
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The
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Fluorescent Probes
Probes and
™
770
IMPORTANT
NOTICE:
The products
described
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coveredare
by covered
one or more
Limited
Use Label
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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
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Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
Section 16.3 Probes for Protein Kinases, Protein Phosphatases
and Nucleotide-Binding Proteins
DiBAC4(5)
The membrane potential–sensing dye bis-(1,3-dibutylbarbituric
acid)pentamethine oxonol (DiBAC 4(5), B436; Figure 16.3.14) initially
inhibits Cl– exchange with an IC50 of 0.146 µM. However, this inhibition
increases with time to an IC50 of 1.05 nM, making DiBAC 4(5) a more
potent inhibitor than DIDS, which has an IC50 of 31 nM under similar
conditions.48
Eosin Maleimide
Although usually selectively reactive with thiols, eosin-5-maleimide (E118, Section 2.2) is known to react with a specific lysine
residue of the band-3 protein in human erythrocytes, inhibiting anion
exchange in these cells and providing a convenient tag for observing
band-3 behavior in the membrane.49–51 Eosin-5-isothiocyanate (E18)
has similar reactivity with band-3 proteins.52,53
Premo™ Halide Sensor
The fluorescent protein–based Premo™ Halide Sensor (P10229)
is a pharmacologically relevant sensor for functional studies of ligand- and voltage-gated chloride channels and their modulators in
cells. Chloride channels are involved in cellular processes as critical
and diverse as transepithelial ion transport, electrical excitability, cell
volume regulation and ion homeostasis. Given their physiological significance, it follows that defects in their activity can have severe implications, including such conditions as cystic fibrosis and neuronal
degeneration. Thus, chloride channels represent important targets for
drug discovery.54 Other methods for detecting chloride are described
in Section 21.2.
Premo™ Halide Sensor combines a Yellow Fluorescent Protein
(YFP) variant sensitive to halide ions with the efficient and noncytopathic BacMam delivery and expression technology (BacMam Gene
Delivery and Expression Technology—Note 11.1). Premo™ Halide
Sensor is based on the Venus variant of Aequorea victoria Green
Resting
Fluorescent Protein (GFP), which displays enhanced fluorescence, increased folding, and reduced maturation time when compared with
YFP. 55 Additional mutations H148Q and I152L were made within the
Venus sequence to increase the sensitivity of the Venus fluorescent
protein to changes in local halide concentration, in particular iodide
ions. 56 Because chloride channels are also permeable to the iodide ion
(I), iodide can be used as a surrogate for chloride. Upon stimulation, a
chloride channel or transporter opens and iodide flows down the concentration gradient into the cells, where it quenches the fluorescence
of the expressed Premo™ Halide Sensor protein (Figure 16.3.15). The
decrease in Premo™ Halide Sensor fluorescence is directly proportional
to the ion flux, and therefore the chloride channel or transporter activity. Premo™ Halide Sensor shows an excitation and emission profile
similar to YFP (Figure 16.3.16) and can be detected using standard
GFP/FITC or YFP filter sets. Halide-sensitive YFP-based constructs in
conjunction with iodide quenching have been used in high-throughput
screening (HTS) to identify modulators of calcium-activated chloride
channels. 57
Premo™ Halide Sensor (P10229) is prepackaged and ready for immediate use. It contains all components required for cellular delivery
and expression, including baculovirus carrying the genetically encoded biosensor, BacMam enhancer and stimulus buffer. Premo™ Halide
Sensor has been demonstrated to transduce multiple cell lines including
BHK, U2OS, HeLa, CHO, and primary human bronchial epithelial cells
(HBEC), providing the flexibility to assay chloride-permeable channels
in a wide range of cellular models. To uncouple cell maintenance and
preparation from cell screening, BacMam-transduced cells can be divided into aliquots and frozen for later assay. Both stable cell lines and
human primary cells can be prepared frozen and “assay-ready” and can
be subsequently plated as little as 4 hours prior to screening. Screening
can be conducted in complete medium and without any wash steps.
Chloride channel assays with Premo™ Halide Sensor are compatible
with standard fluorescence HTS platforms.
Activated
Iodide
Closed
Ion channel
Open
250
200
150
100
50
0
Extracellular
Intracellular
Extracellular
Intracellular
Figure 16.3.15 Principle of Premo™ Halide Sensor Sensor (P10229): Iodide redistribution
upon chloride channel activation. Basal fluorescence from Premo™ Halide Sensor is high
when chloride channels are closed or blocked. Upon activation (opening) of chloride channels, the iodide ions enter the cell, down its concentration gradient, and quench the fluorescence from Premo™ Halide Sensor.
B
NaCl 0 mM
NaCl 100 mM
NaCl 500 mM
Fluorescence emission
(arbitrary units)
Ion channel
A
Fluorescence emission
(arbitrary units)
Premo™
Halide Sensor
500
550
600
Wavelength (nm)
650
200
Nal 0 mM
Nal 20 mM
Nal 60 mM
Nal 100 mM
Nal 300 mM
Nal 500 mM
150
100
50
0
500
550
600
650
Wavelength (nm)
Figure 16.3.16 Quenching of Premo™ Halide Sensor fluorescence by increasing concentrations of iodide and chloride. U2OS cells were transduced with Premo™ Halide Sensor.
After 24 hours, cells were trypsinized and lysed by resuspension in sterile distilled water.
Fluorescence quenching of the lysate was examined using increasing concentrations of NaCl
(A) and NaI (B). Iodide induces substantially greater quenching of Premo™ Halide Sensor fluorescence than chloride.
™
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 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
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771
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
Section 16.3 Probes for Protein Kinases, Protein Phosphatases
and Nucleotide-Binding Proteins
REFERENCES
1. J Physiol (2004) 555:251; 2. Neurosci Lett (2004) 358:75; 3. Proc Natl Acad Sci U S A
(1992) 89:3586; 4. J Cell Biochem (2007) 100:86; 5. Biochemistry (2006) 45:8940;
6. Pharm Res (2003) 20:537; 7. Biochem Pharmacol (2004) 67:285; 8. J Histochem
Cytochem (2002) 50:731; 9. Mol Pharmacol (1991) 40:490; 10. Am J Physiol (1993)
264:C1577; 11. J Physiol (1999) 515 (Pt 1):109; 12. Cell Calcium (1997) 22:99;
13. Proc Natl Acad Sci U S A (2004) 101:10554; 14. Nature (1997) 388:882; 15. J Biol
Chem (1983) 258:3503; 16. J Med Chem (1967) 10:66; 17. J Membr Biol (1988) 105:1;
18. Biochimie (1988) 70:1285; 19. Mol Pharmacol (1986) 30:112; 20. Biochemistry
(1984) 23:4481; 21. J Biol Chem (1984) 259:4313; 22. Mol Pharmacol (1984) 25:131;
23. Biochemistry (1977) 16:531; 24. J Biol Chem (1998) 273:28813; 25. Cell Biol Int
(1994) 18:723; 26. Physiol Res (1994) 43:33; 27. Biochemistry (1986) 25:8133; 28. J Biol
Chem (1985) 260:14484; 29. J Biol Chem (1982) 257:5601; 30. Biochemistry (1998)
37:6658; 31. Biophys Chem (1998) 71:245; 32. Cell Tissue Res (1990) 260:529; 33. J
Cell Biol (1986) 103:1473; 34. J Biol Chem (1984) 259:11176; 35. Biochemistry (1980)
19:969; 36. Biophys J (2008) 94:2654; 37. Drug Discov Today (2007) 12:396; 38. Trends
Pharmacol Sci (1990) 11:417; 39. Diabetes (1999) 48:2390; 40. Pflugers Arch (1997)
434:712; 41. J Biol Chem (2001) 276:33369; 42. Anal Biochem (2009) 394:30; 43. Assay
Drug Dev Technol (2008) 6:765; 44. J Biol Chem (2009) 284:14020; 45. Am J Physiol
(1992) 262:C803; 46. J Biol Chem (1994) 269:21489; 47. J Biol Chem (1991) 266:13355;
48. Am J Physiol (1995) 269:C1073; 49. Biochemistry (1995) 34:4880; 50. Biophys
J (1994) 66:1726; 51. Am J Physiol (1993) 264:C1144; 52. Biochim Biophys Acta
(1987) 897:14; 53. Biochim Biophys Acta (1979) 550:328; 54. Nat Rev Drug Discov
(2009) 8:153; 55. Nat Biotechnol (2002) 20:87; 56. FEBS Lett (2001) 499:220; 57. Mol
Pharmacol (2008) 73:758.
DATA TABLE 16.3 PROBES FOR ION CHANNELS AND CARRIERS
Cat. No.
MW
Storage
Soluble
Abs
EC
Em
A1322
788.89
F,D,L
DMSO
362
7500
471
B436
542.67
L
DMSO, EtOH
590
160,000
616
B7431
769.18
F,D,L
DMSO, EtOH
504
74,000
511
B23461
858.74
F,D,L
DMSO
503
80,000
510
C22803
873.05
F,D
DMSO
<300
none
341
61,000
415
D337
498.47
F,DD
H2O
286
41,000
none
D338
500.48
F,DD
H2O
352
32,000
none
D673
474.32
L
H2O
D7443
686.48
F,D,L,A
DMSO, EtOH
504
83,000
511
E18
704.97
F,DD,L
pH >6, DMF
521
95,000
544
378
23,000
423
E3111
336.22
D,L
H2O, MeOH
587
60,000
615
E34250
915.23
F,D,L
DMSO, H2O
504
76,000
511
E34251
783.10
F,D,L
DMSO, H2O
S7445
760.57
F,D,L,A
DMSO, EtOH
565
143,000
570
For definitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.
Notes
1. Oxonols may require addition of a base to be soluble.
2. Isothiocyanates are unstable in water and should not be stored in aqueous solution.
Solvent
MeOH
MeOH
MeOH
MeOH
Notes
H2O
MeOH
H2O
MeOH
pH 9
MeOH
MeOH
MeOH
MeOH
2
2
PRODUCT LIST 16.3 PROBES FOR ION CHANNELS AND CARRIERS
Cat. No.
Product
A1322
B10334
B10146
B10331
B10332
B10333
B10147
B10341
B10335
B10019
B10033
B436
B23461
B7431
C22803
D337
D338
D7443
D673
E18
E34251
E34250
E3111
F10016
F10017
P10020
P36207
P36208
P10229
S7445
9-anthroyl ouabain
BacMam Kir1.1 *for 10 microplates*
BacMam Kir2.1 *for 10 microplates*
BacMam Kv1.1 *for 10 microplates*
BacMam Kv1.3 *for 10 microplates*
BacMam Kv2.1 *for 10 microplates*
BacMam Kv7.2 and Kv7.3 *for 10 microplates*
BacMam Nav1.2 *for 10 microplates*
BacMam Nav1.5 *for 10 microplates*
BacMam-hERG *for 10 microplates*
BacMam-hERG *for 100 microplates*
bis-(1,3-dibutylbarbituric acid)pentamethine oxonol (DiBAC4(5))
BODIPY® FL ouabain
BODIPY® FL verapamil, hydrochloride
CEDA, SE (5-(and-6)-carboxyeosin diacetate, succinimidyl ester) *mixed isomers*
DIDS (4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid, disodium salt)
4,4’-diisothiocyanatodihydrostilbene-2,2’-disulfonic acid, disodium salt (H2DIDS)
DM-BODIPY® (–)-dihydropyridine *high affinity enantiomer*
DNDS (4,4’-dinitrostilbene-2,2’-disulfonic acid, disodium salt)
eosin-5-isothiocyanate
ER-Tracker™ Green (BODIPY® FL glibenclamide) *for live-cell imaging*
ER-Tracker™ Red (BODIPY® TR glibenclamide) *for live-cell imaging*
5-(N-ethyl-N-isopropyl)amiloride, hydrochloride
FluxOR™ Potassium Ion Channel Assay *for 10 microplates*
FluxOR™ Potassium Ion Channel Assay *for 100 microplates*
PowerLoad™ concentrate, 100X
Premo™ Cameleon Calcium Sensor *for 10 microplates*
Premo™ Cameleon Calcium Sensor *for 100 microplates*
Premo™ Halide Sensor *for 10 microplates*
ST-BODIPY® (-)-dihydropyridine *high affinity enantiomer*
TheMolecular
MolecularProbes®
Probes Handbook:
Handbook: AAGuide
and Labeling
LabelingTechnologies
Technologies
The
Guideto
toFluorescent
Fluorescent Probes
Probes and
™
772
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
1
2
Quantity
5 mg
1 kit
1 kit
1 kit
1 kit
1 kit
1 kit
1 kit
1 kit
1 kit
1 kit
25 mg
100 µg
1 mg
5 mg
100 mg
100 mg
25 µg
1g
100 mg
100 µg
100 µg
5 mg
1 kit
1 kit
5 mL
1 kit
1 kit
1 kit
25 µg