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Polychromatic Flow Cytometry and Flow Applications

Polychromatic Flow Cytometry and Flow Applications. Basic Concepts Kevin P. Weller Technical Applications Specialist. CD45. CD4/CD8. CD28. recognition. CD3. CD3. Lck. β. α. γ. ε. δ. ε. ITAM. FYN. signaling. TCR. ECM. ζ ζ. T Cell. APC. CD45. CD4/CD8. MHC Class II.

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Polychromatic Flow Cytometry and Flow Applications

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  1. Polychromatic Flow Cytometry and Flow Applications Basic Concepts Kevin P. Weller Technical Applications Specialist

  2. CD45 CD4/CD8 CD28 recognition CD3 CD3 Lck β α γ ε δ ε ITAM FYN signaling TCR ECM ζζ T Cell

  3. APC CD45 CD4/CD8 MHC Class II Cytoskeletal Changes CD28 Lck β α Jun Fos γ ε δ ε FYN Zap 70 LAT Grb2 Ras NF-AT, PKC,Ras SOS Gads PLCg1 ECM ζζ IL-2 T Cell Co-stimulation MAPK promotor

  4. Polychromatic Flow Cytometry • What Do We Need For PFC? • Chemistry – the fluorescent dyes • Must be bright (S/N) • Minimal spectral overlap • Straightforward conjugation to antibodies • Instrumentation • More Light Sources: Multi-laser (2- 4 or more) • More Detectors: 6 to 16 or more parameters • More Efficient Optical Pathway: Higher sensitivity • Higher resolution & fast complex data handling :Digital Electronics & New Graphical User Interface

  5. Polychromatic Flow Cytometry Practical Considerations: Improving Resolution • Experimental design: • Make “Good” Fluorochrome/Antigen Density Choices/Matches • Implementing multicolor panels is principally empirical and requires many iterations (time) • Optimize Instrument Setup • Photomultiplier Voltages • Compensation/Spillover • Add necessary controls • Fluorescence Minus One (FMO) • Autofluorescence Controls • Optimized Isotypic Controls • Hardware • Digital Electronics: Eliminate Analog Artifacts • Software: • Automate compensation • Enhance graphical user interface

  6. “Bright” = good resolution sensitivity D W1 W2

  7. Various fluorochromes-stain index

  8. Best color Combinations…

  9. Building An PCF Assay: Relative Antigen Densities • Approximate Relative Antigen Densities from Technical Data Sheets • Match The Lowest Density Antigen To The Brightest Fluorochrome, etc…………. • Limited by conjugate availability • New cheaper custom conjugates available • Consider potential spectral overlap

  10. Building An PCF Assay:Laser Choices Optimize Your PCF Assay By: • Using Multiple Laser Lines • Don’t “Pack” A Laser Line • Choose “Optimal” Laser/Fluorochrome Combinations: • To Minimize Spillover Background • To Optimize Signal :Noise • Optimize Filter Choices to Minimize Spillover • Use JAVA Applet on BD Website

  11. Building An PCF Assay:Effect of Spillover on Double Stained Cells Compensated analog data: CD45 PerCP allows same dim CD4 cells to be separated from bkg. – little spillover into PE CD45- PerCP Dim CD4-PE Compensated analog data: CD45 FITC makes dim CD4 difficult to measure due to FITC spillover into PE and resultant “spread” CD45-FITC Dim CD4-PE

  12. Top 4 Sources of Problems In Multi-Color Analyses 1.) Compensation 2.) Compensation 3.) Compensation 4.) Compensation

  13. Basic Principles of Compensation - The Problem FL1 = FITC + x% PE FL2 = PE + y% FITC Remember the basic assumption of flow analysis: The signal in FL1= the signal from FITC and only FITC and the signal in FL2= the signal from PE and only PE. This is NOT TRUE for the raw data! The process by which each fluorescence channel is “corrected” for this spectral overlap is termed Fluorescence Compensation

  14. Compensation - Too Little; Too Much

  15. Compensation- Too Dim, Too Bright Compensation Controls Small errors in compensation of a dim control (A) can result in large compensation errors with bright reagents (B & C).

  16. Building An PCF Assay:Spillover increases background http://www.bdbiosciences.com/spectra/

  17. Building An PCF Assay:FMO (Fluorescence Minus One) • Compensated data exhibits spread • Bright single positives may change threshold levels between dim and background in other dimensions • Use where autofluorescence and/or isotypic controls are NOT useful for determining threshold over background • The best control is one stained with all reagents except the one of interest

  18. Building An PCF Assay:FMO (Fluorescence Minus One) PBMC were stained as shown in a 4-color experiment. Compensation was properly set for all spillovers Courtesy Mario Roederer

  19. Setting Up For A PCF Assay: Compensation: BDTM CompBeads • Three Specificities • Anti-mouse Ig, kappa • Anti-rat Ig, kappa • Anti-rat/hamster Ig, kappa • Negative Control Bead • Supplied in sets: Positive & Negative Bead • Stain with reagents used for PCF Assay • Optimal Spillover Control • 50% positive/50% negative Control

  20. Setting Up For A PCF Assay: Compensation: BDTM CompBeads Method: For Each Conjugate: • Add 1 drop (60 ul) of positive bead and 1 drop of negative bead to 100 ul of staining buffer in a tube or well • Add optimally titered antibody • Incubate 15-30 minutes RT • Wash with staining buffer • Resuspend pellet in staining buffer • Run according to instructions for automated spillover algorithm

  21. Setting Up For A PCF Assay: Compensation: BDTM CompBeads FITC Spillover calculation AutoCompensation method Matrix algebra (PE = 0.83%) ±183.8 2010 2010 Spillover coefficient = slope ±33.7 5 5 1. 7981 7981 10 10 2. 3. 4.

  22. Setting Up For A PCF Assay: Compensation: CD20 BDTM CompBeads Populations are aligned In dye space PEc = PE x 1.00209 + FITC x -0.25203 Not a subtraction, rather a correction because we use matrix algebra and compensation coefficients. ±133.6 ±33.6

  23. Setting Up For A PCF Assay:Compensation: Tandems CD8 CD3 Time Sample Left in Light PE-Cy7 PE-Cy5 0 hours PE (FL2) 2 hours 22.5 hours

  24. Logicle: Compensated Biexponential Display Log at the upper end, linear at the low, and symmetrical about zero. Biexponential transform where data zero is shown by the crosshairs in the plot • This FlowJo example shows the value of a mostly logarithmic scale on the upper end, and a lower linear region occupies a reasonable plot area compared to that in the blended scale. • Compensated single pos are continuous • All populations are visible

  25. PCF: Questions of T Cell Differentiation Questions of T Cell Differentiation that can be Addressed with Polychromatic Flow Cytometry • What is the CD45RA/CD27/CD28 phenotype of antigen-specific CD4 and CD8 T cells? • In IFNg+ versus IL-2+ cells? • In CMV- versus HIV-specific cells? • In CMV-specific cells of HIV- versus HIV+ donors?

  26. 8-color antigen-specific immunophenotyping

  27. 8 Color Compensation (LSR II) Single-stained controls: CD4 FITC CD4 PE CD28 PerCP-Cy5.5 CD45RA PE-Cy7 Auto- comp CD27 APC CD8 APC-Cy7 CD3 Pacific Blue CD4 AmCyan Spillover Matrix

  28. Hierarchal Gating Strategy

  29. Phenotype of CMV-responsive CD4 T cells IFNg Response IL-2 Response 1 0 1 1 CD45RA 29 78 69 20 68 64 29 20 CD28 0 3 14 1 CD27

  30. Phenotype of CMV-responsive CD8 T cells IFNg Response IL-2 Response 1 1 8 3 CD45RA 20 78 58 30 3 21 52 7 CD28 15 36 26 40 CD27

  31. CFC Staining Protocol - Key Steps Stimulate and Harvest Cells Block Fc Receptors Stain Cell Surface Antigens Fix and Permeabilize Cells Stain Intracellular Cytokines Intracellular Staining Controls Analyze by Flow Cytometry

  32. T Cell Immunology Tool Kit Traditional Cytokine Flow Cytometry IL-2 Phycoerythrin CD4 FITC

  33. CFC & Proliferation BrdU Flow Kit Measures cellular incorporation of BrdU with gentle fixation and permeablization at neutral pH which allows the concomitant detection of other cellular determinants. • Bromodeoxyuridine (BrdU) is a thymidine analog • Allows measurement of cell proliferation and cell cycle status • May be used in vitro and in vivo • BrdU is incorporated into the DNA of cycling cells • Incorporated BrdU is detected with anti-BrdU mAb • Prolonged exposure identifies cycling cells • Pulse labeling allows determination of cell-cycle kinetics

  34. CFC & Proliferation • Allows the correlation of: • Phenotype • Cytokine expression • Cell Cycle • Proliferation

  35. T Cell Immunology Tool Kit Antigen Specific Cytokine Flow Cytometry CD69 PE anti-TNF FITC

  36. Antigen Specific Cytokine Flow Cytometry • Simultaneous single cell detection of cell surface and intracellular events (e.g. cytokines, activation antigens, proliferation, phenotypic markers) • Whole blood (physiological conditions) • Rapid method (<6 h) • Compatible with variety of stimuli including antigen

  37. CD4+ T cell cytokine response to HIV-1 antigen following 3 immunizations with Remmune Viral load: <400 CD4: 1147 CD69-PE anti-IFNg FITC

  38. What is a Cytometric Bead Array (CBA) = ELISA

  39. Bead-based Immunoassays Capture Ab Capture Bead + + Analyte of Interest Fluorescent Detector Ab

  40. Beads and Flow Cytometry –A Powerful Tool 104 IL-8 103 IL-6 102 + Bead Intensity FL3 (670LP) 101 100 100 101 102 103 104 Detector Ab Intensity FL2 (585/42BP)

  41. New CBA Flex Beads 1 2 3 4 5 6 7 8 9 A B C D E F G H I • Single Size: • Forward/Side Scatter: 7um size, 99% singlets • Maintain PE Reporter system with excitationoff 488 or 532 source • Indexing options • Systems with 2 channels off 635nm • BD FACSArray • BD FACSAria • BD FACSCanto • BD LSRII • Systems with 1 channel on 635nm • BD FACSCalibur

  42. Plex definition: clustering Double clickor drag to assign

  43. 9-Plex for Measuring T Cell Activation 1 6 1. Itk (Y511) 2. ERK (T202/Y204) 3. JNK (T183/Y185) 4. P38 (T180/Y182) 5. PLCg (Y783) 6. ZAP70 (Y319) 7. LAT (Y171) 8. c-Jun (S63) 9. RSK (S380) 2 3 4 5 7 9 8

  44. Phagocytosis: Fluorescent Beads Quantitative Phagocytosis using fluorescent beads

  45. Activation: Calcium Flux 390 nm/ 495 nm Ratio Of Ca++ Bound Indo-1 at 390 nm to Free Indo-1 at 495 nm

  46. Cytotoxicity: NK Mediated PKH-26 Labeled NK Sensitive YAC-1 Lymphoma Cell Line NK Effector Cells To-Pro-3 DNA Dye http://sci.cancerresearchuk.org/axp/facs/davies/ISACXXI.pdf

  47. P-Glycoprotein: Drug Resistance P-glycoprotein is a transmembrane protein that acts as an ATP-dependent efflux pump. This efflux activity has been suggested to lead to resistance to the drugs used in chemotherapy

  48. pH: pH Dependent GFP Changes in Mitochondrial-Matrix pH Induced by Ultraviolet Radiation

  49. Signal Transduction:Fluorescence Resonance EnergyTransfer Interaction between Fas and FADD death Domains visualized by FRET Fas CFP Plasmids were cotransfected with YFP expression vector YFP-C1 (Clontech) or A vector encoding FADD (DD)-YFP The upper right quadrants indicate the % of CFP positive cells exhibiting FRET

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