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Multicolor Flow Cytometry Workshop

Multicolor Flow Cytometry Workshop. Holden Maecker, PhD. Learning Objectives. Explain the critical aspects of digital and multicolor flow cytometry that make it different from traditional analog flow cytometry with 1 – 4 colors

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Multicolor Flow Cytometry Workshop

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  1. Multicolor Flow Cytometry Workshop Holden Maecker, PhD

  2. Learning Objectives • Explain the critical aspects of digital and multicolor flow cytometry that make it different from traditional analog flow cytometry with 1–4 colors • Describe the role of instrument configuration in the performance of multicolor flow cytometry • Perform instrument QC using BDTM Cytometer Setup and Tracking beads, and understand the use of baseline and application settings in BD FACSDivaTM software • Design a robust multicolor reagent panel, understanding the role of spillover, tandem dyes, and antibody titration • Create appropriate controls for a multicolor experiment, and be able to find and correct potential problems in multicolor data

  3. Schedule: Day 1 9:00–10:30 I. Introduction, review of basic concepts 10:30–10:45 Break 10:45–11:45 II. Digital and multicolor flow cytometry Exercise 1: Adjusting biexponential displays 12:00–1:00 Lunch 1:00–2:00 III. Instrument setup, optimization, and QC Exercise 2: Determining stain index and spill index 2:00–4:00 Acquisition of data: • Instrument characterization using CS&T • Baseline and application settings determination • Compensation using BD™ CompBeads • 8-color stained PBMCs

  4. Schedule: Day 2 9:00–10:00IV. Design and optimization of multicolor panels: • Selection of fluorochromes • Matching fluorochromes with antibody specificities • Determining application-specific settings Demonstration: Visualizing data on a virtual cytometer 10:00–12:00 Data analysis in BD FACSDiva software 12:00–1:00 Lunch 1:00–2:30 V. Controls and Data QC Exercise 3: Finding and correcting a spillover problem 2:30–2:45 Break 2:45–4:00 Review and summary, discussion of participant issues

  5. Multicolor Flow Cytometry Workshop:I. Review of Flow Cytometry Basics

  6. Outline • Definitions, what can be measured by flow cytometry • Fluidics: Sheath and sample streams, flow cells, sorting • Optics: Lasers, filters • Electronics: PMTs, signal processing • Fluorochromes: Spectra, spillover • Data analysis: FCS files, gating, statistics

  7. Definitions • Flow cytometry: The study of cells as they move in fluid suspension, allowing multiple measurements to be made for each cell. • FACSTM: Fluorescence-activated cell sorting

  8. What Measurements Can Be Made? • Forward scatter (FSC): Proportional to cell size • Side scatter (SSC): Proportional to cell granularity • Fluorescence: • Binding of fluorescent-labeled antibodies • Ca++-sensitive dyes within cells • Fluorescent proteins expressed by cells • Binding of DNA dyes

  9. Largestand most granular population 1000 Granulocytes 800 600 Side Scatter 400 Monocytes 200 Smallestand least granular population Lymphocytes 0 0 200 400 600 800 1000 Forward Scatter Scatter Profile of Lysed Whole Blood

  10. Fluorescence Data Display Negative control histogram PE Number of Events FITC Fluorescent Intensity  FITC

  11. Major Components of a Flow Cytometer • Sample injection port • Sheath and waste reservoirs • Flow cell • Laser(s) • Optical filters • Photomultiplier tubes ( PMTs ) or photodiodes • Signal processor

  12. Cytometer Fluidics Create Laminar Flow Sample stream Flow cell Sheath stream Laser beam Cell

  13. Cell Sorting

  14. MulticolorExperiment Cytometer Configuration Longpass filter Bandpass filter PMT

  15. Background and Autofluorescence All cells have a certain level of background fluorescence due to: • Autofluorescence from pigments and fluorescent moieties on cellular proteins • Non-specifically bound antibodies and free antibody in the sample stream The level of autofluorescence varies with the wavelength of excitation and collection: • Highest in FITC, PE detectors • Lowest in far red (APC, Cy™7) detectors

  16. Fluorescence Sensitivity Detection Efficiency (Q): The number of photoelectrons generated per molecule of fluorophore • Dependent upon fluorophore, laser, filters, PMT sensitivity, voltage gain setting, etc. Background (B): Non-specific signal intrinsic to the system • Dependent upon autofluorescence, unbound fluorophore, ambient light, etc.

  17. Common Fluorophores for Ab Conjugation

  18. Fluorescence Spillover Emission of FITC in the PE channel

  19. 1,650 – 185 • 3,540 – 125 Compensating for Spillover Compensated Uncompensated FITC mean fluorescence PE mean fluorescence ---------------------------- ---------------------------- Negative Positive Negative Positive ----------- ---------- ----------- ---------- Uncompensated 125 3,540 185 1,650 Compensated 125 3,560 135135 % Spillover = X 100

  20. FCS Files • FCS 2.0 and FCS 3.0 conventions • Often referred to as list-mode files • Contain all of the measurements (FSC-H, FSC-A, SSC-H, SSC-A, FL1-H…) for each individual cell processed in a given sample

  21. Hierarchical Gating

  22. Web Reference Tools • BD Spectrum Viewer: www.bdbiosciences.com/spectra • Maecker lab weblog: http://maeckerlab.typepad.com (protocols, manuscripts, literature updates)

  23. Multicolor Flow Cytometry Workshop:II. Digital & Multicolor Flow Cytometry

  24. Differences From Analog Instruments • Optics: Fiber optics and octagons/trigons • Fluidics: Optimized for high flow rates • Electronics: Digital signal processing

  25. PMT Octagon and Trigon PMT Bandpass filter Longpass filter

  26. Filter Nomenclature Conventions Longpass (LP) filter: Allows light above a certain wavelength to pass, reflects shorter wavelengths • Example: 505 LP = 505 nm longpass Bandpass (BP) filter: Allows light within a certain range of wavelengths to pass (above and below a specified midpoint) • Example: 530/30 BP = 515–545 nm bandpass

  27. Effect of Flow Rate • Higher flow rates mean a broader sample stream ( less precise focusing) • Less precise focusing means less accurate fluorescence measurement of dim populations ( population spreading) • Higher flow rates also increase the frequency of coincident events (can be gated out based on FSC area vs height) • In practice, flow rates of 8,000–12,000 events per second are acceptable on the BD™ LSRII (vs 2,000–3,000 events per second on a BD FACSCalibur™ flow cytometer)

  28. Digital Signal Processing Generates high resolution fluorescence values that can include negative numbers • No compression of populations at the low end of the fluorescence scale • More accurate representation of dim populations Allows compensation to be performed in the software at any time • Uncompensated data and any associated compensation matrix are both stored • Compensation can be changed at any time Peak area and peak height can both be recorded for all parameters

  29. Biexponential Display of Digital Data Antibody capture beads stained with 3 levels of an APC reagent The transformed display shows aligned populations In the APC-Cy7 dimension APC-Cy7 Area All populations align correctly APC Area

  30. Spillover Affects Resolution Sensitivity Without CD45 AmCyan With CD45 AmCyan CD19 FITC

  31. Conclusions • Optical platforms using octagons and trigons result in more efficient light collection and flexibility in the use of detectors and filters • BD LSR II fluidics allow running at higher flow rates with minimal compromise to the data • Digital signal processing provides more accurate representation of dim populations, and more accurate and flexible compensation—but logarithmic data display may not be appropriate • More colors mean more spillover issues, with loss of resolution sensitivity in affected detectors

  32. Exercise 1 Adjusting biexponential displays: • Open the FCS file “exercise1.fcs” • Gate on small lymphocytes, then on double-positive events for CD45 AmCyan vs CD3 Pacific Blue • From this gate, create a plot of CD4 FITC vs CD8 APC-H7 • Turn on biexponential scaling for the x- and y-axes, and note the changes to the plots • Turn on manual biexponential scaling and experiment with various scaling factors for FITC and APC-H7, noting how the plots change Questions: • Would gating be affected by biexponential scaling? • Is it important to use the same scaling for all samples in an experiment?

  33. Multicolor Flow Cytometry Workshop:III. Instrument Setup and QC

  34. Outline • Configure your instrument • Characterize your instrument • Design your panel • Optimize settings for your panel • Run appropriate controls • QC your data

  35. Outline • Configure your instrument • Number and type of lasers • Number of PMTs per laser • Choice of filters and dichroic mirrors These choices will determine: • What fluorochromes you can use effectively • How well certain fluorochrome combinations will perform

  36. W1 W2 How Do We Measure Performance? Resolution Sensitivity D Stain Index = D / W Where D = difference between positive and negative peak medians W = 2 x rSD (robust standard deviation)

  37. CD127 PE 40 35 30 25 mW green laser (532 nm) 25 100 mW blue laser (488 nm) Stain index 20 25 mW blue laser (488 nm) 15 10 5 0 300 400 500 600 700 PMT voltage An Example: Green vs Blue Lasers • Green laser is more efficient for PE and PE tandems • Blue laser is more efficient for FITC, PerCP, and GFP

  38. Second Example: Filters and Spillover

  39. Outline • Characterize your instrument • Obtain minimum baseline PMT settings • Track performance over time This allows you to: • Run the instrument where it is most sensitive • Be alert to changes in the instrument that might affect performance

  40. Automated Baseline PMT Voltage Determination Using BD CS&T Baseline PMTV is set by placing the dim bead MFI to equal 10X SDEN 460 V SDEN = 20 MFI= 200

  41. FITC Channel (Blue laser) 550 525 500 PMT Voltage 475 450 425 400 10/22/04 11/11/04 12/01/04 12/21/04 01/10/05 01/30/05 02/19/05 03/11/05 Time Performance Tracking A variety of parameters can be tracked: • Linearity, CVs, laser alignment • PMT voltages must hit target values Data can be visualized in Levey-Jennings plots:

  42. Exercise 2 Calculating stain index and spill index: • Open the FCS file “exercise2.fcs” (AmCyan Compbeads) • Calculate the stain index in the primary detector (AmCyan) by determining: [Median (positive peak)] - [Median (neg peak)] 2 x rSD (neg peak) • Calculate the spill index in FITC by determining the FITC stain index as above, then calculating: [Stain index (FITC) / Stain index (AmCyan)] Questions: • What is an acceptable stain index? • How high can the spill index be before it is problematic?

  43. Stain Index for Various Fluorochromes

  44. Antibody Cocktail for Data Acquisition • CD4 FITC • CD127 PE • HLA-DR PerCP-Cy™5.5 • CD45RA PE-Cy7 • CD25 APC • CD8 APC-H7 • CD3 V450 • CD45 AmCyan

  45. Schedule: Day 2 9:00–10:00IV. Design and optimization of multicolor panels: • Selection of fluorochromes • Matching fluorochromes with antibody specificities • Determining application-specific settings Demonstration: Visualizing data on a virtual cytometer 10:00–12:00 Data analysis in BD FACSDiva software 12:00–1:00 Lunch 1:00–2:30 V. Controls and Data QC Exercise 3: Finding and correcting a spillover problem 2:30–2:45 Break 2:45–4:00 Review and summary, discussion of participant issues

  46. Multicolor Flow Cytometry Workshop:IV. Panel Design & Application Settings

  47. Outline • Design your panel • Reserve the brightest fluorochromes for the dimmest markers and vice versa • Avoid spillover from bright populations into detectors requiring high sensitivity • Beware of tandem dye issues • Titrate antibodies for best separation This allows you to: • Maintain resolution sensitivity where you need it most • Avoid artifacts of tandem dye degradation

  48. Various Fluorochromes—Stain Index

  49. Spillover Affects Resolution Sensitivity Without CD45 AmCyan With CD45 AmCyan CD19 FITC Note that this is only an issue when the two markers (CD45 and CD19) are co-expressed on the same cell population.

  50. Special Requirements for Tandem Dyes Compensation requirements for tandem dye conjugates can vary, even between two experiments with the same antibody • Degrade with exposure to light, temperature, and fixation • Stained cells are most vulnerable Solutions: • Minimize exposure to above agents • Use BD stabilizing fixative if a final fix is necessary • Run label-specific compensation

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