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Novel Flow Cytometry Reagents from Molecular Probes® Jolene Bradford R&D Associate Director, Flow Cytometry Systems June 2013 Research Use Only. Not for use in diagnostic procedures. Cell Health, Stress, & Death Are my cells alive? • Cell viability & vitality Are my cells performing basic functions? Are my cells dividing and proliferating? •Cell morphology •Cell motility •Endocytosis/Phagocytosis •Transcription •Protein/gene expression •Signal transduction •Ion homeostasis •Cell proliferation •Cell cycle Are my cells healthy or stressed? •Oxidative stress/damage •Apoptosis •Autophagy Cell Health, Stress, & Death Are my cells alive? • Cell viability & vitality Are my cells performing basic functions? Are my cells dividing and proliferating? •Cell morphology •Cell motility •Endocytosis/Phagocytosis •Transcription •Protein/gene expression •Signal transduction •Ion homeostasis •Cell proliferation •Cell cycle Are my cells healthy or stressed? •Oxidative stress/damage •Apoptosis •Autophagy Viability & Vitality Cells exist anywhere on a continuum between healthy and dead Live Proliferation Membrane Potential Dead Enzyme Activity Apoptosis Autophagy Membrane Integrity Viability detection – Easy additions Typically measures membrane integrity Traditional reagents (Cell‐impermeant DNA dyes) − Fixable reagents (LIVE/DEAD® Fixable dyes—also called amine reactive dyes) − Eliminate dead cells from analysis B A Live + Dead Cells Live Cells Viability: Impermeant nucleic acid‐binding dyes Viability: Integrity of plasma membrane Cytosol Nucleus + SYTOX® Red Stain Nonviable (Dead) Viable (Live) Impermeant Nucleic Acid Dyes, Flow Cytometry Dyes which penetrate cells with a compromised cell membrane to stain nucleic acids, but do not cross the membranes of live cells > Can be used to identify dead cells in a population > Can be used to quantitate DNA content in fixed cells Propidium Iodide (488 nm ex) 7‐AAD (488 nm ex) SYTOX® AADvanced™ dead cell stain (488 nm ex) SYTOX® Green dead cell stain (488 nm ex) SYTOX® Orange dead cell stain (488 /532/561 ex) SYTOX® Blue dead cell stain (405 nm ex) SYTOX® Red dead cell stain (633 nm ex) SYTOX® Dead Cell Stains Five different colors for flexibility in multicolor panels Propidium Iodide ReadyProbes® Reagent – power and simplicity • Ready-to-use liquid propidium iodide formulation • Rapid staining of dead cells without wash steps • Ready-to-use liquid formulation in convenient dropper bottle—no need to dilute, weigh, or pipette • Stable at room temperature—keep handy at your work station or cell culture area EL4 cells labeled with Propidium Iodide Data collected on the EVOS® FLoid ® Imaging Station during the 36th Annual Research Course in Flow Cytometry held at University of New Mexico, 2013 LIVE/DEAD® Fixable Dead Cell Stains Live Dead Before fixation Live Dead After fixation Live cells: react with the kit’s fluorescent reactive dye only on their surface to yield weakly fluorescent cells. Live Dead Cells with compromised membranes: react with the dye throughout their volume, yielding brightly stained cells. Viability = membrane integrity Fixable violet dead cell stain 405 nm Violet Excitation (440/40 BP) Effect of fixation on dead cell dyes Before Fixation After Fixation Propidium iodide 488 nm excitation, 610/20 filter Before Fixation After Fixation LIVE/DEAD® Fixable Red stain 488 nm excitation, 610/20 filter LIVE/DEAD® Fixable Dead Cell Stain Kits Rigorous precautions for analysis of biohazardous specimens Fixation procedures that produce minimal distortion of their characteristics are highly advantageous Compatible with Fix & perm procedures Advantages of Kit Reduced handling risks‐ formaldehydes are known to reduce risks of viruses Specificity and reliability –staining pattern is similar before and after fixation. Convenience‐Cells can be stained and fixed at various times during the experiment, and the results can be analyzed several hours later, without loss of the discrimination pattern. Compatible with Fix and Perm Procedures ‐useful for dead cell identification with intracellular targets LIVE/DEAD® Fixable Dead Cell Stains: amine-reactive dyes Before Fixation 18 Hours Post-Fixation LIVE/DEAD® Fixable Blue stain Before Fixation 18 Hours Post-Fixation LIVE/DEAD® Fixable Green stain 488 nm excitation, 530/30 filter UV (355 nm) excitation, 450/50 filter LIVE/DEAD® Fixable Violet stain LIVE/DEAD® Fixable Red stain 405 nm excitation, 450/20 filter LIVE/DEAD® Fixable Aqua stain 488 nm excitation, 610/20 filter LIVE/DEAD® Fixable Far Red stain 633 nm excitation, 660/20 filter 405 nm excitation, 530/30 filter LIVE/DEAD® Fixable Yellow stain LIVE/DEAD® Fixable Near IR stain 405 nm excitation, 575/25 filter 633 nm excitation, 780/60 filter Tips & Tricks • Impermeant DNA Dyes • Add at final step, do not wash out • Emission is broad, consider for multicolor applications • Dead/Fixed cells can be used for compensation control • Amine‐reactive Dyes • Do not use protein in buffers • Live cells have dim fluorescence • Use with ‐aldehyde fixatives • Can be used without fixing cells too • ArC™ compensation beads useful Vitality Functionality of metabolic or enzymatic processes Cytosol Nucleus + Calcein violet Nonvital (Dead) Vital (Live) Probing enzyme activity with Calcein AM • Esterase substrates – Electrically neutral – Freely diffuse into cells – Nonspecific intracellular esterases convert to fluorescent products • Live cells – Bright fluorescence – Retained in cells Live and ethanol-killed bovine pulmonary artery epithelial cells (BPAEC) stained with calcein AM. Live cells with active enzymes fluoresce bright green. Probing enzyme activity Kit Ex max Em max Calcein Green AM 495 nm 515 nm Calcein Blue AM 360 nm 449 nm Calcein Violet AM 400 nm 452 nm Calcein Red‐Orange AM 577 nm 590 nm Live bovine pulmonary artery endothelial cells (BPAEC) were incubated simultaneously with calcein red-orange AM and MitoTracker® Green FM and NucBLue. Vitality: measure of metabolic activity Calcein AM 405 nm + 633 nm Excitation SYTOX red fluorescence 488 nm + 633 nm Excitation Calcein fluorescence Calcein violet fluorescence Calcein AM - 488 nm ex -Violet 405 nm ex -Blue UV ex 405 nm Excitation Vitality: measure of metabolic activity: C12‐Resazurin C12-Resazurin -Blue 488 nm ex Reactive Oxygen Species (ROS) Why is oxidative stress important? Reactive Oxygen Species (ROS) form as a natural byproduct of normal metabolism of oxygen, important role in homeostasis and cell signaling. atherosclerosis Increased oxidative stress has been implicated in: Atherosclerosis Neurodegenerative disorders Diabetes and cardiovascular diseases Cancer Chronic liver diseases Lung diseases α-Synuclein (Parkinson’s) CellROX® Green Flow Cytometry Assay Kit *for oxidative stress detection* Uses the FITC detection channel BPAE CellROX® Green Reagent (green); Hoechst 33342 (blue) Compatible with fixation Number of Cells Counted TBHP 50‐ 200µM control CellROX® Green Reagent Fluorescence CellROX® Deep Red Flow Cytometry Assay Kit *for oxidative stress detection* Uses the APC detection channel CellROX® Deep Red Reagent control U2OS U2‐OS CellROX® Deep Red Reagent (purple); CellMask™ Orange plasma membrane stain (orange); SYTO® Green fluorescent nuclear stain (green) Compatible with fixation TBHP + 1mM NAC TBHP CellROX® Orange Flow Cytometry Assay Kit *for oxidative stress detection* TBHP + 1mM NAC U2‐OS control CellROX® Orange Reagent fluorescence Compatible yellow or green laser excitation TBHP Apoptosis Vitality/Viability Continuum Apoptosis LIVE DEAD Apoptosis is a type of cell death Apoptosis (αποπτοσισ) Apo = off, away ‐ptosis = a falling Greek, apoptosis translates to “falling off" of petals from flowers, or leaves from plants or trees. -A carefully regulated process that is part of normal development and homeostasis. -Dysregulation of apoptosis is implicated in disease states such as cancer, autoimmune disease and degenerative conditions. Apoptosis Ca2+ Chemical or γ Irradiation O2 Calpain Activation Ca2+ O2 Bcl-2 dATP pCasp-9 Apoptosome APAF-1 APAF-1 pCasp-9 Cyto c NAD Cyto c ↓ΔΨm Energy Cyto c Effector Caspase PARP Caspase-3 Caspase-6 Caspase-7 Mitochondria Free Radicals EndoG AIF Caspase-9 DNA Fragmentation Apoptosis DNA Damage Necrosis Relative Timeframe – Jurkat Model Function Reagent Relative Time Live Mitochondria Activity changes Membrane potential Transition pore Dead MitoTracker® Red dye DiOC2(3), DiIC1(5), JC-1 MTP assay PS translocation, Annexin V, F2N12S Membrane Permeability YO-YRO™1, PO-PRO™-1 Caspase activity Cell Event™ Capsase Green, Caspase substrates Metabolic activity C12 resazurin, Calcein AM Nuclear condensation Hoechst, DyeCycle™ Violet dye Sub G0 peak DyeCycle™ Orange dye Membrane integrity PI, SYTOX® dead cell stains * Jurkat cells induced with 10 µM camptothecin EVOS® Imaging System Smarter systems | Easier cell imaging | Faster results Key Features Application Description XL Core XL Advanced Basic transmitted transmitted light light digital digital inverted inverted system system Routine cell culture In‐hood applications FLoid FL/FL Color FL Auto Basic fluorescence system Advanced fluorescence system Fully automated fluorescence system Versatile light Cell Culture Requiring Advanced automated microscopy needs Fluorescence Advanced fluorescence imaging Chromogenic stains Quick view of cells, imaging Time lapse applications, (ICC, IHC), tissue fluorescent labeling Highly configurable multiwell plate scanning culture and teaching and image stitching/ tiling •Advanced •21” monitor software •Ergonomic Design •Time‐lapse •Simplified user interface •Minimal learning curve •Up to 5 objectives •Up to 4 fluor. Channels •Time‐lapse imaging •Capture color images •Plate scanning •Obj./Filter flexibility EL4 control cells bright field image Data collected on the EVOS® FLoid ® Imaging Station during the 36th Annual Research Course in Flow Cytometry held at University of New Mexico, 2013 Treated EL4 cells bright field image Data collected on the EVOS® FLoid ® Imaging Station during the 36th Annual Research Course in Flow Cytometry held at University of New Mexico, 2013 MitoProbe™ Reagents: Detecting Mitochondrial Changes Ca2+ Chemical or γ Irradiation O2 Calpain Activation Ca2+ O2 Bcl-2 dATP pCasp-9 Apoptosome APAF-1 APAF-1 pCasp-9 Cyto c NAD Cyto c ↓ΔΨm Energy Cyto c Effector Caspase PARP Caspase-3 Caspase-6 Caspase-7 Mitochondria Free Radicals EndoG AIF Caspase-9 MitoTracker® and MitoProbe™ Assay Kits DNA Fragmentation MitoTracker® Red Dye − MitoProbe™ JC-1 Assay Kit − MitoProbe™ DiIC1(5) Assay Kit − MitoProbe™ Transition Pore Assay Kit DNA Damage − Apoptosis Apoptosis Assay Kits Necrosis Carbocyanine Dyes: MitoProbe™ JC‐1 dye 3T3 cells JC-1 red fluorescence Untreated Jurkat JC-1 JC-1 green fluorescence JC-1 red fluorescence Camptothecin Treated Jurkat JC-1 green fluorescence 2 μM JC-1 in Jurkat 488 nm Excitation a a a a a a a a a a a a a a a a a a a a JC-1 on BPAE Cells Carbocyanine Dyes: MitoProbe™ DiIC1(5) dye DiIC1(5) on MRC5 cells Events Treated DiIC1(5) 633 nm Excitation 50 nM DiIC1(5) Healthy Carbocyanine Dyes: MitoProbe™ DiIC1(5) dye - MitoProbe™ DiIC1(5) - Caspase 3 - 7-AAD dead cell stain Data from Bill Telford, NCI (NIH) MitoTracker® Red CMXRos Dye MitoTracker® RedCMXRos dye Uses the PE detection channel Healthy Treated APC-annexin V 488 nm & 633 nm Excitation 40 nM MitoTracker® Red CMXRos Mitochondrial Membrane Potential/Annexin V Apoptosis Kit: MitoTracker® Red and Alexa Fluor® 488 annexin V control Annexin V treated Annexin V Loss of Membrane Asymmetry / Loss of Integrity Ca2+ Annexin V Conjugates − − − − − − − − − − Ca2+ Fluor® Chemical or γ Irradiation Alexa 350 (346/442) Pacific Blue™ (410/455) dATP Alexa Fluor® 488 (495/519) pCasp-9 Cyto c Fluorescein (496/519) APAF-1 Cyto c Apoptosome APAF-1 R-phycoerythrin (496/575) Cyto c pCasp-9 Alexa Fluor® 568 (578/603) Effector Caspase-3 Caspase-6 ® Caspase Alexa Fluor 594 (590/617) Caspase-7 Alexa Fluor® 647 (650/668) Caspase-9 Allophycocyanin (650/660) Biotin Monomeric Cyanines O2 Calpain Activation DNA Fragmentation Bcl-2 PARP O2 NAD ↓ΔΨm Energy Cell Impermeant Nucleic Acid Stains EndoG Mitochondria 7-aminoactinomycin D (7-AAD) Free Radicals − Propidium Iodide (PI) AIF − SYTOX® dead cell dyes − LIVE/DEAD® Fixable Dead Cell Stains DNA Damage - Membrane permeability Ratiometric Membrane Asymmetry - F2N12S Apoptosis Necrosis Loss of Membrane Asymmetry: Annexin V Control Treated TIPS for Annexin V assays Calcium and Magnesium are required for annexin V binding to PS; binding is reversible, so divalent cations must be present during the entire assay Analysis should be carried out quickly following labeling Some cells (e.g. megakarocytes, platelets, some myeloid lineage cells, microvesicles) may have large amounts of PS on their surface In advanced apoptotic or necrotic cells, annexin V can label the inner membrane leaflet Adherent cells removed by mechanical scraping or trypsin may flip their PS residues independent of apoptosis; careful removal is required Monomeric Cyanine Dyes Membrane Permeability/Dead Cell Apoptosis Kits with YO‐PRO™‐1 and PO‐PRO™‐1 dyes D L A YO-PROTM-1 fluorescence 488 nm excitation 488 nm and 405 nm excitations Violet Ratiometric Membrane Asymmetry Probe 4'‐N,N‐diethylamino‐6‐(N,N,N‐dodecyl‐methylamino‐sulfopropyl)‐methyl‐3‐hydroxyflavone F2N12S Normal cells exhibit an asymmetry in lipid distribution between the outer and inner cell membranes with phosphatidyl‐serine (PS) and phosphatidylethanolamine (PE) normally located on the inner leaflet of the cell membrane During apoptosis PS and PE translocate from the inner to outer leaflet of the cell membrane, changing the surface charge of the outer leaflet. This translocation facilitates recognition and elimination of these cells by macrophages. The Violet Ratiometric Membrane Asymmetry Probe, F2N12S, is a novel violet excitable dye for the detection of changes in membrane asymmetry by detecting variations in surface charge, and gives two emission bands. F2N12S Membrane Asymmetry Probe a Ratiometric dye is self calibrating Independent of cell size, cell concentration and instrument variation 5 minute labeling time, no wash No special buffer required Use with adherent cells also Dual labeling of F2N12S vs SYTOX® AADvanced™ stain ratio of 405ex with 530/30 and 603/48 ratio emission orange fluorescence VL3 1 SYTOX® AADvanced™ fluorescence green fluorescence VL2 1 control Ratio fluorescence of VL3/VL2 1 orange fluorescence VL3 1 SYTOX® AADvanced™ fluorescence green fluorescence VL2 1 apoptotic Ratio fluorescence of VL3/VL2 Caspase Assay Kits Ca2+ Chemical or γ Irradiation O2 Calpain Activation Ca2+ O2 Bcl-2 dATP pCasp-9 Apoptosome APAF-1 APAF-1 pCasp-9 Cyto c NAD Cyto c ↓ΔΨm Energy Cyto c Effector Caspase PARP Mitochondria Caspase-3 Caspase-6 Caspase-7 Free Radicals EndoG AIF Caspase-9 Caspase Activity DNA Fragmentation Apoptosis Damage CellEvent™ 3/7 Caspase GreenDNA Reagent Vybrant® FAM Caspase-3 and –7 Assay Kit Vybrant® FAM Caspase-8 Assay Kit Vybrant® FAM Polycaspases Assay Kit Necrosis CellEvent™ Caspase 3/7 Green Reagent A nucleic acid dye conjugated to DEVD peptide Add CellEvent™ reagent ÆFluorogenic Caspase 3/7 Substrate Active caspase 3/7cleaves the DEVD peptide and the free nucleic acid dye binds to DNA. ADVANTAGES: − Live cell amenable, no-wash protocol − May be added to complete growth media − Retained after fixation and permeabilization − May be multiplexed with other live or fixed cell probes Incubate 30 min Read Active Caspase-3/7 Enzyme DEVD DNA dye Non-fluorescent No DNA binding Bound DNA dye CellEvent® Caspase 3/7 Green Detection Reagent Control cells Apoptosis-induced cells Caspase 3/7 + U2‐OS EL4 control cells labeled with CellEvent™ Caspase 3/7 Green Data collected on the EVOS® FLoid ® Imaging Station during the 36th Annual Research Course in Flow Cytometry held at University of New Mexico, 2013 Treated EL4 cells labeled with CellEvent™ Caspase 3/7 Green Data collected on the EVOS® FLoid ® Imaging Station during the 36th Annual Research Course in Flow Cytometry held at University of New Mexico, 2013 EL4 control cells labeled with CellEvent™ Caspase 3/7 Green and NucBlue™ LIVE Data collected on the EVOS® FLoid ® Imaging Station during the 36th Annual Research Course in Flow Cytometry held at University of New Mexico, 2013 Treated EL4 cells labeled with CellEvent™ Caspase 3/7 Green and NucBlue™ LIVE Data collected on the EVOS® FLoid ® Imaging Station during the 36th Annual Research Course in Flow Cytometry held at University of New Mexico, 2013 CellEvent™ Caspase‐3/7 Green Detection Reagent for the detection of activated caspase 3/7 -A four amino peptide (DEVD) conjugated to a nucleic acid binding dye -Cell Permeant, intrinsically non-fluorescent -With activation of caspase-3 or caspase-7 the DEVD peptide is cleaved, enabling the dye to bind to DNA producing a green fluorescence Monitor Cell Health using CellROX® Deep Red and CellEvent ® Caspase 3/7 Green Detection Reagents Increased oxidative stress Caspase 3/7 activation HeLa HeLa CellROX® Deep Red Reagent (purple); CellEvent® Caspase‐3/7 Green Detection Reagent (green); Hoechst 33342 (blue) Multiplexing Apoptosis Assays: CellEvent® Caspase 3/7 Green Fluorescence SYTOX® AADvanced™ Fluorescence SYTOX® AADvanced™ Fluorescence Annexin V Pacific Blue® Fluorescence •CellEvent® Caspase 3/7 Green Detection Reagent •Annexin V Pacific Blue™ conjugate •SYTOX® AADvanced™ Dead Cell Stain Annexin V Pacific Blue® Fluorescence CellEvent® Caspase 3/7 Green Fluorescence Mitochondrial Health and Apoptosis: TMRM with CellEvent™ Caspase 3/7 Green Tetramethylrhodamine methyl ester 0 hour 1.7 hours 2.8 hours 3.3 hours 4.0 hours 4.7 hours 5.3 hours 6.2 hours 7 hours HeLa Cells treated with 0.5 µM staurosporine Immunodetection of cleaved caspase 3 (PE conjugate) and LIVE/DEAD® Fixable Near-IR labeling Since caspase 3 immunolabeling requires fixation and permeablization, combine it with a LIVE/DEAD® Fixable Dead cell Stain. Cells should be labeled with the Live/Dead reagent prior to fixation. camptothecin 5 μM 16 h Near IR Live/Dead viability untreated “late” apoptotic “early” apoptotic “viable” cells PE anti-cleaved caspase 3 Data from Bill Telford, NCI (NIH) Detecting Nuclear Changes Ca2+ Chemical or γ Irradiation O2 Calpain Activation Ca2+ O2 Bcl-2 dATP pCasp-9 Apoptosome APAF-1 APAF-1 pCasp-9 Cyto c NAD Cyto c ↓ΔΨm Energy Cyto c Effector Caspase PARP Caspase-3 Caspase-6 Caspase-7 Mitochondria Free Radicals EndoG AIF Caspase-9 Nuclear Condensation − DNA Fragmentation DNA Damage Apoptosis Kits Sub-G1 Population − DyeCycle™ Orange Dye DNA Fragmentation − Click-iT® TUNEL Assay Kit Apoptosis Necrosis Nuclear Condensation Propidium iodide fluorescence UV & 488 nm Excitation 405 nm & 488 nm Excitation 405 nm & 488 nm Excitation Untreated Jurkat cells Propidium iodide fluorescence Hoechst 33342 fluorescence Camptothecin-induced Jurkat cells Condensed Chromatin Signal Hoechst 33342 fluorescence Hoechst 33342 + propidium iodide DyeCycle™ Violet dye + SYTOX® AADVanced™ stain Developing Apoptotic Controls • General drug treatments – Staurosprine/camptothecin/valinomycin – Concentration/time course varies from cell – Understand cell model • Dead Cell Control – Good to have! – Heat Killing – Alcohol Killing (cells can be stored for years) 3 day Hands-on Apoptosis Workshop Held July 16-18, $750 Life Technologies Training Facility in Frederick, MD This research methods course will focus on using the flow cytometry platform to learn about apoptosis as measured with both common and cutting-edge assays. This course is aimed at individuals with beginner to intermediate experience, and consists of a series of seminars and hands-on laboratory sessions. The seminar portion of the workshop will include a brief introduction to the basics of flow cytometry and in-depth discussions of methodologies for studying apoptosis. Laboratory sessions will include sample preparation, flow cytometer instrument operation, data collection, data analysis, and troubleshooting tips. A basic fluorescent imaging system will also be available to visualize cells. http://www.learn.lifetechnologies.com/courses/view/id/345 Proliferation: DNA Content Cell Cycle What is cell cycle? Cell cycle describes the progression of a cell through a cycle of division. DNA content distribution Number of cells G0/G1 G0/G1 G2M S 2N Linear scale G2M S 4N Dye fluorescence A single time point measurement shows cells in different phases of the cycle, with distribution in three major phases of the cell cycle. Under ideal staining conditions, all G1 or G2 cells are expected to be uniform in staining. However in practice, the cell populations are represented on frequency histograms with peaks of various widths. Frequency Histogram showing DNA content distribution Live Jurkat cells stained with Hoechst 33342 Frequency distribution histogram & software deconvolution Cell‐Permeant Nucleic Acid Dyes Dyes which have the ability to penetrate an intact cell membrane to stain nucleic acid These dyes can be used for determining the DNA content of viable cells. Allows resolution of cell cycle information against the dynamic background of LIVING cells − Hoechst dyes (UV ex) dsDNA(A‐T) − Vybrant® DyeCycle™ Violet stain (UV, 405 ex) dsDNA − Vybrant® DyeCycle™ Green stain (488 ex) dsDNA − Vybrant® DyeCycle™ Orange stain (488 & 532 ex) dsDNA − Vybrant® DyeCycle™ Ruby stain (488–633 ex) dsDNA Sorting: Vybrant® DyeCycle™ Orange stain Vybrant® DyeCycle™ Orange stain: sorting of NIH 3T3 cells Post-sort verification of populations G0G1: G2M: 3 days post sort 3 days post sort Simple Protocols 1.1 Remove the Vybrant® DyeCycle™ Violet stain from the refrigerator and allow it to equilibrate to room temperature. 1.2 Prepare flow cytometry tubes each containing 1 mL of cell suspension in complete media at a concentration of 1 × 106 cells/mL. 1.3 To each tube add 1 μL of Vybrant® DyeCycle™ Violet stain and mix. 1.4 Incubate at 37˚C for 30 minutes, protected from light. 1.5 Analyze without washing on a flow cytometer using ~405 nm excitation and ~440 nm emission. Technical Considerations for Live Cell Cycle Instrument − Know your instrument (lasers/emission filters) − Know your dye (excitation/emission) and read product information − Proper maintenance and careful optical alignment − Verify instrument linearity Sample Prep − Single cell suspension − Cell concentration and dye concentration − Optimize for cell type, medium or buffer used, time of incubation, temperature of incubation. Acquisition and Analysis − Acquire sample in low flow rate with traditional hydrodynamic focusing systems − Any collection rate works with acoustic focusing systems − Total number of cells acquired − Gating strategies and software analysis FxCycle™ Stains: for DNA content measurements in fixed cells Enables multicolor experiments utilizing DNA content measurements Count FxCycleTM Violet stain HL-60 promyeloblast FxCycleTM Violet FxCycleTM Far Red stain TF-1 erythroblast FxCycleTM Far Red FxCycleTM PI/RNase stain Jurkat TLymphoblast FxCycleTM PI/RNase Proliferation: Dye Dilution Cell Proliferation Analysis by Dye Dilution Cell division results in equal partitioning of dye between daughter cells. Fluorescence of daughter cells is half that of parent cell Fourth Generation Third Generation First Generation Number of Cells Second Generation Brightness CellTrace™ Experimental Protocol 20µL DMSO 1. Bring a vial of CellTrace™ dye to room temperature. DMSO CellTrace™ (Dry) Dissolve 2. Add 20µL anhydrous DMSO to prepare a 5mM stock solution. 3. Add 1µL of stock solution to 1mL cells for a final concentration of 5µM. 4. Incubate 30 minutes. 5. Quench and wash. 6. Proceed with stimulation and analysis. 5mM CellTrace™ in DMSO 1µL dye into 1mL cells Incubate 30min Quench and wash Stimulate and analyze CellTrace™ CSFE Multicolor and Gating CellTrace™ CFSE: Occupies a Popular Channel CellTrace™ Violet: Generational Analysis CellTrace™ Violet Analyzed with Proliferation Modeling Software CellTrace™ Violet Multicolor and Gating Cellular Lights® Talin GFP + CellTrace™ Violet stain U2OS human osteosarcoma cells transduced with Cellular Lights® Talin-GFP and stained 20 minutes at room temperature with a 5µM solution of CellTrace™ Violet in phosphatebuffered saline. Proliferation: Click-iT® EdU Frequency Histogram DNA content distribution Frequency Histogram DNA content distribution A549 lung cancer cells Etoposide treated Where is the S-phase? G1: 21.98 % G2: 62.92 % S: 15.10 % DNA content Nocodazole treated G1: 1.77 % G2: 82.74 % S: 15.49 % DNA content Thymidine Analogs 3H-thymidine Radioactive Cannot multiplex BrdU Requires DNA denaturation for detection with antibody Cell cycle stains require dsDNA No DNA denaturation required for detection EdU Multiplex compatible – including antibodies and stains for cell cycle analysis Just what is “click” chemistry? Click chemistry describes a set of chemical reactions for use in chemical library synthesis. However the name has stuck to one conjugation reaction in particular: Copper catalyzed azide-alkyne cycloaddition N N N + N - C R” + Azide C Cu+ R’ Alkyne N N C C R’ Triazole The reaction is efficient, rapid, stable and bio-orthogonal R” “click” chemistry and cell proliferation This concept can be applied to the labeling and detection of DNA, using a thymidine analog containing a terminal alkyne group and a dye-labeled azide. Click chemistry-based labeling and detection Copper catalyzed azide-alkyne cycloaddition Azide Triazole Alexa Fluor® 488 Dye Cu(I) ++ DNA Alkyne Room Temp DNA BrdU Br Br Br BrdU (5-bromo-2’-deoxyuridine) Br Incoroprated BrdU is inaccessible to the BrdU antibody in dsDNA Br Br Br Br •BrdU antibody requires DNA denaturation for detection •Numerous protocols: acid, heat, or nuclease for DNA denaturation Br Br Br Br Br Br Br Denatured DNA is required for antibody detection of BrdU Br EdU EdU (5-ethynyl-2’-deoxyuridine) Click-iT® EdU detection Click labeling does not require DNA denaturation Dye azide reacts with the alkyne on double stranded DNA Click-iT® EdU cell proliferation : Flow Cytometry Simplified Workflow Click-iT® EdU follows a basic protocol of aldehyde fixation and detergent permeabilization Fix for 15 minutes, wash Permeabilize for 30 minutes, wash Incubate in click labeling mixture for 30 minutes, wash Optional: Incubate with cell cycle stain for 15-30 minutes Analyze Attune® Acoustic Cytometer with Click-iT ® EdU Alexa Fluor® 488 azide and FxCycle™ Violet Collected at Standard EdU- Alexa Fluorr® 488 fluorescence EdU- Alexa Fluor ® 488 fluorescence 100 µl/min FxCycle™ Violet fluorescence FxCycle™ Violet fluorescence Number of Cells Click-iT™ EdU Alexa Fluor® 647 fluorescence Attune® Acoustic Cytometer with Click-iT® EdU Alexa Fluor® 647 azide and propidium iodide Click-iT® EdU Alexa Fluor® 647 fluorescence Propidium iodide fluorescence Click-iT™ EdU cell proliferation : Flow Cytometry Workflow Variations LIVE/DEAD® Fixable Dead Cell stain, wash Surface immunostaining, wash Fix for 15 minutes, wash Permeabilization for 30 min, wash Click label and Perm together 30 min, wash Incubate in click labeling cocktail for 30 minutes, wash Intracellular immunostaining, wash Optional: Cell Cycle stain for DNA content 15-30 min Analyze LIVE/DEAD® Fixable Stains: amine reactive dyes can be used with aldehyde fixation Click-iT® EdU kit can be used with the amine reactive dyes Violet-fluorescent reactive dye vs SSC Live cells Live & Dead cells Click‐iT™ EdU detection reagents: Flow Cytometry Pacific Blue™ azide Violet laser Alexa Fluor® 488 azide Blue laser Alexa Fluor® 647 azide Red laser Click-iT ® EdU Cell Proliferation Kits Available Flow Cytometry: Click-iT® Alexa Fluor ® 488 azide Click-iT ® Pacific Blue ® azide Click-iT ® Alexa Fluor ® 647 azide High-Throughput Imaging (HCS) : Click-iT ® Alexa Fluor ® 488 azide Click-iT ® Alexa Fluor ® 594 azide Click-iT ® Alexa Fluor ® 647 azide Imaging: Click-iT ® Alexa Fluor ® 488 azide Click-iT ® Alexa Fluor ® 594 azide Click-iT ® Alexa Fluor ® 647 azide Pacific Green™ dye: Expanded Options for the Violet laser Number of Cells Number of Cells excitation max 411 nm; emission max 500 nm CD3-biotin + Streptavidin-Pacific Green™ Secondary CD3 + Zenon®-Pacific Green™ Mouse IgG1 complex Number of Cells Number of Cells CD3 –Pacific Green™ Direct Conjugate CD3 + Goat anti-Mouse Pacific Green™ Secondary Flow cytometry mobile app Cell Imaging mobile app 3D Cell mobile app Flow Cytometry Resource Center New Fluorescence Spectraviewer Webinars on Flow and Imaging Cytometry Share & Learn on Facebook Thank You! © 2013 Life Technologies Corporation All rights reserved. Research Use Only. Not for use in diagnostic procedures.
