Polychromatic Flow Cytometry and Flow Applications Basic Concepts



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Polychromatic Flow Cytometry and Flow Applications

  • Basic Concepts
  • Kevin P. Weller
  • Technical Applications Specialist
  • T Cell
  • ζ ζ
  • TCR
  • recognition
  • signaling
  • CD3
  • CD3
  • ITAM
  • ECM
  • FYN
  • CD45
  • CD4/CD8
  • Lck
  • α
  • β
  • ε
  • ε
  • δ
  • γ
  • CD28
  • T Cell
  • ζ ζ
  • ECM
  • FYN
  • CD45
  • CD4/CD8
  • Zap 70
  • α
  • β
  • ε
  • ε
  • δ
  • γ
  • APC
  • MHC Class II
  • CD28
  • Lck
  • Co-stimulation
  • LAT
  • Gads
  • SOS
  • Grb2
  • PLCg1
  • Ras
  • MAPK
  • Cytoskeletal Changes
  • promotor
  • Jun
  • Fos
  • NF-AT, PKC,Ras
  • IL-2

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

Polychromatic Flow Cytometry

  • 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:
  • Practical Considerations: Improving Resolution

“Bright” = good resolution sensitivity

  • W2
  • W1
  • D

Various fluorochromes-stain index

  • Reagent
  • Clone
  • Filter
  • Stain Index
  • PE
  • RPA-T4
  • 585/40
  • 356.3
  • Alexa 647
  • RPA-T4
  • 660/20
  • 313.1
  • APC
  • RPA-T4
  • 660/20
  • 279.2
  • PE-Cy7
  • RPA-T4
  • 780/60
  • 278.5
  • PE-Cy5
  • RPA-T4
  • 695/40
  • 222.1
  • PerCP-Cy5.5
  • Leu-3a
  • 695/40
  • 92.7
  • PE-Alexa 610
  • RPA-T4
  • 610/20
  • 80.4
  • Alexa 488
  • RPA-T4
  • 530/30
  • 75.4
  • FITC
  • RPA-T4
  • 530/30
  • 68.9
  • PerCP
  • Leu-3a
  • 695/40
  • 64.4
  • APC-Cy7
  • RPA-T4
  • 7801/60
  • 42.2
  • Alexa 700
  • RPA-T4
  • 720/45
  • 39.9
  • Pacific Blue
  • RPA-T4
  • 440/40
  • 22.5
  • AmCyan
  • RPA-T4
  • 525/50
  • 20.2

Best color Combinations…

  • 6-color
  • 8-color
  • 10-color
  • Additional
  • FITC or Alexa 488
  • FITC or Alexa 488
  • FITC or Alexa 488
  • FITC or Alexa 488
  • PE
  • PE
  • PE
  • PE
  • PE-Texas Red or PE-Alexa 610
  • PE-Texas Red or PE-Alexa 610
  • PerCP-Cy5.5
  • PerCP-Cy5.5
  • PerCP-Cy5.5
  • PerCP-Cy5.5
  • PE-Cy7
  • PE-Cy7
  • PE-Cy7
  • PE-Cy7
  • APC or Alexa 647
  • APC or Alexa 647
  • APC or Alexa 647
  • APC or Alexa 647
  • Alexa 680 or 700
  • Alexa 680 or 700
  • APC-Cy7
  • APC-Cy7
  • APC-Cy7
  • APC-Cy7
  • AmCyan
  • AmCyan
  • AmCyan
  • Pacific Blue
  • Pacific Blue
  • Pacific Blue
  • Q-dot 655, 705…

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

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

Building An PCF Assay: Effect of Spillover on Double Stained Cells

  • CD45-FITC
  • Dim CD4-PE
  • Compensated analog data:
  • CD45 FITC makes
  • dim CD4 difficult to measure
  • due to FITC spillover into
  • PE and resultant “spread”
  • CD45- PerCP
  • Dim CD4-PE
  • Compensated analog data:
  • CD45 PerCP allows
  • same dim CD4 cells to be
  • separated from bkg. – little
  • spillover into PE
  • 1.) Compensation
  • 2.) Compensation
  • 3.) Compensation
  • 4.) Compensation

Basic Principles of Compensation - The Problem

  • 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
  • FL1 = FITC + x% PE
  • FL2 = PE + y% FITC

Compensation - Too Little; Too Much

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).

Building An PCF Assay: Spillover increases background

  • http://www.bdbiosciences.com/spectra/

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

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

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

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

Setting Up For A PCF Assay: Compensation: BDTM CompBeads

  • 2010
  • 5
  • 7981
  • 10
  • Spillover coefficient = slope
  • FITC Spillover calculation
  • AutoCompensation method
  • Matrix algebra (PE = 0.83%)
  • 2010
  • 5
  • 7981
  • 10
  • 1.
  • 2.
  • 3.
  • 4.
  • ±183.8
  • ±33.7

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

Setting Up For A PCF Assay: Compensation: Tandems

  • 0 hours
  • 2 hours
  • 22.5 hours
  • PE
  • (FL2)
  • CD8
  • CD3
  • PE-Cy5
  • PE-Cy7
  • Time Sample Left in Light

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

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 IFN+ versus IL-2+ cells?
      • In CMV- versus HIV-specific cells?
      • In CMV-specific cells of HIV- versus HIV+ donors?

8-color antigen-specific immunophenotyping

  • Ab Conjugate
  • Laser 
  • CD28 PerCP-Cy5.5
  • 488
  • CD45RA PE-Cy7
  • 488
  • CD27 APC
  • 633
  • Surface
  • CD8 APC-Cy7
  • 633
  • staining
  • CD3 Pacific Blue
  • 405
  • CD4 AmCyan
  • 405
  • Anti-IFN FITC
  • 488
  • Intracellular
  • Anti-IL-2 PE
  • 488
  • staining

8 Color Compensation (LSR II)

  • CD4 FITC
  • CD4 PE
  • CD28 PerCP-Cy5.5
  • CD45RA PE-Cy7
  • CD3 Pacific Blue
  • CD4 AmCyan
  • CD27 APC
  • CD8 APC-Cy7
  • Single-stained controls:
  • Auto-
  • comp
  • Spillover Matrix

Hierarchal Gating Strategy

Phenotype of CMV-responsive CD4 T cells

  • CD27
  • IFN Response
  • IL-2 Response
  • CD28
  • CD45RA
  • 0
  • 1
  • 78
  • 20
  • 1
  • 1
  • 69
  • 29
  • 64
  • 20
  • 14
  • 1
  • 68
  • 29
  • 3
  • 0

Phenotype of CMV-responsive CD8 T cells

  • IFN Response
  • IL-2 Response
  • CD27
  • CD28
  • CD45RA
  • 8
  • 3
  • 30
  • 58
  • 1
  • 1
  • 20
  • 78
  • 3
  • 21
  • 36
  • 40
  • 7
  • 52
  • 15
  • 26

CFC Staining Protocol - Key Steps

  • Stimulate and Harvest Cells
  • Stain Cell Surface Antigens
  • Stain Intracellular Cytokines
  • Intracellular Staining Controls
  • Analyze by Flow Cytometry
  • Block Fc Receptors

T Cell Immunology Tool Kit

  • Traditional
  • Cytokine Flow Cytometry
  • IL-2 Phycoerythrin
  • CD4 FITC

CFC & Proliferation

  • 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
  • BrdU Flow Kit

CFC & Proliferation

  • Allows the correlation of:
  • Phenotype
  • Cytokine expression
  • Cell Cycle
  • Proliferation

T Cell Immunology Tool Kit

  • Antigen Specific
  • Cytokine Flow Cytometry
  • CD69 PE
  • anti-TNF FITC

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

What is a Cytometric Bead Array (CBA)

  • ELISA
  • =

Bead-based Immunoassays

  • +
  • +
  • Analyte of Interest
  • Fluorescent Detector Ab
  • Capture Bead
  • Capture Ab

Beads and Flow Cytometry – A Powerful Tool

  • +
  • IL-8
  • IL-6
  • 100
  • 100
  • 101
  • 101
  • 102
  • 102
  • 103
  • 103
  • 104
  • 104
  • Bead Intensity
  • FL3 (670LP)
  • Detector Ab Intensity
  • FL2 (585/42BP)

New CBA Flex Beads

  • Single Size:
    • Forward/Side Scatter: 7um size, 99% singlets
  • Maintain PE Reporter system with excitation off 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
  • A
  • B
  • C
  • D
  • E
  • F
  • G
  • H
  • I
  • 1
  • 2
  • 3
  • 4
  • 5
  • 6
  • 7
  • 8
  • 9

Plex definition: clustering

  • Double click or drag to assign

9-Plex for Measuring T Cell Activation

  • 1
  • 2
  • 3
  • 4
  • 5
  • 6
  • 7
  • 8
  • 9
  • 1. Itk (Y511)
  • 2. ERK (T202/Y204)
  • 3. JNK (T183/Y185)
  • 4. P38 (T180/Y182)
  • 5. PLC (Y783)
  • 6. ZAP70 (Y319)
  • 7. LAT (Y171)
  • 8. c-Jun (S63)
  • 9. RSK (S380)

Phagocytosis: Fluorescent Beads

  • Quantitative
  • Phagocytosis
  • using
  • fluorescent
  • beads

Activation: Calcium Flux

  • 390 nm/ 495 nm
  • Ratio Of Ca++
  • Bound Indo-1
  • at 390 nm to
  • Free Indo-1
  • at 495 nm

Cytotoxicity: NK Mediated

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

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

pH: pH Dependent GFP

  • Changes in
  • Mitochondrial-Matrix
  • pH Induced by
  • Ultraviolet Radiation

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

Cell Division: CSFE

  • 5-(and-6)-carboxyfluorescein
  • diacetate, succinimidyl ester
  • is a lipophilic dye which
  • incorporates into the cell
  • membrane
  • The amount of dye in the cell
  • Membrane of proliferating cells
  • halves with each successive
  • division

Übersicht Apoptose

Apoptosis: Mitochondrial Membrane Potential

  • Control
  • Staurosporine 1m 4hrs
  • FL1-H
  • FL1-H
  • FL2-H
  • FL2-H

Apoptosis: Quantitative Analysis of Caspase-3 Activation

  • Jurkat cells Treated with Campothecin
  • Caspase 3 PE

Apoptosis: A Time Course

  • Annexin
  • PI

Apoptosis: PARP Cleavage

  • Poly-ADP Ribose Polymerase
  • Is Cleaved By Caspases During
  • Apoptosis.
  • HeLa Cells Treated With 4 m
  • Campothecin for 4 hours
  • Staining with anti-Cleaved PARP FITC
  • Indicates 40% Apoptotic Index

Apoptosis: TUNEL Assay

  • Jurkat Cells Treated for 6 hours with IgM Anti Fas Antibody
  • lysis
  • Y
  • Z
  • Phospho-
  • specific
  • Ab blot
  • Limited opportunity to view variability
  • Limited statistics
  • Requires sorting of subsets to gain access to intracellular antigens (not easily multiplexed)
  • Requires large #s of cells (106)
  • Lysates: not living cells
  • 0.1
  • 1
  • 10
  • 100
  • 1000
  • Flow
  • cytometry
  • 1
  • 110
  • 1100
  • 4671
  • 115
  • 25
  • 50
  • 7
  • 227
  • Current methods for assessing phosphorylation
  • Possible to observe heterogeneity
  • Considerably enhanced statistics
  • Can subset via surface markers to gain access to rare cell types
  • Requires fewer cells (103 - 104)
  • Simultaneous detection of multiple post-translational modifications within heterogenic cell populations

11 Color Flow Cytometry Looking at Signaling in Subpopulations of Cells

  • Perez & Nolan, Nature Biotechnology, Vol 20, p155-162
  • Multi-Dimensional Analysis

Development of Visualization tools

  • Nolan et al.

Systems Biology : Signaling Network Mapping

  • Kinase 1
  • Kinase 11
  • Kinase 2
  • Kinase 6
  • Kinase 3
  • Kinase 1
  • Kinase 3
  • Kinase 1
  • Kinase 11
  • Kinase 4
  • Kinase 9
  • Kinase 11
  • Kinase 11
  • Surface 2
  • p-p44/42
  • p-p38
  • CD28
  • CD27
  • P-JNK
  • CD45RA
  • CD62L
  • CD11A
  • CD8
  • CD3
  • CD4
  • 0
  • 0
  • 0
  • 1651
  • 1914
  • 2594
  • 2180
  • 2746
  • 2506
  • 0
  • 0
  • 1443
  • 1382
  • 451
  • 1887
  • 2088
  • 1992
  • 0
  • 2149
  • 1674
  • 1631
  • 1722
  • 1755
  • 0
  • 1575
  • 2311
  • 0
  • 1821
  • 0
  • 2427
  • 1280
  • 1835
  • 1592
  • 1629
  • 1621
  • 1290
  • 2429
  • 0
  • 1008
  • 0
  • 2023
  • 1686
  • 2348
  • 1208
  • 0
  • 1600
  • 0
  • 1767
  • 0
  • 2496
  • 0
  • 2412
  • 939
  • 0
  • 733
  • 1671
  • 0
  • 2409
  • 2203
  • 0
  • 2141
  • 0
  • 1981
  • 1012
  • 2750
  • 2016
  • 0
  • 0
  • 0
  • 0
  • 0
  • 2029
  • 0
  • 2201
  • 2466
  • 2486
  • 1136
  • 0
  • 0
  • 0
  • 0
  • 0
  • 2230
  • 0
  • 2272
  • 2619
  • 2471
  • 1023
  • 2034
  • 0
  • 2439
  • 2021
  • 2397
  • 0
  • 1611
  • 2033
  • 1300
  • 2642
  • 2255
  • 0
  • 2367
  • 0
  • 0
  • 0
  • 2650
  • 2731
  • 1669
  • 0
  • 2166
  • 1252
  • 1750
  • 1373
  • 1780
  • 2253
  • 1441
  • 0
  • 1448
  • 2109
  • 1688
  • 2338
  • 1777
  • 786
  • 1344
  • 0
  • 2168
  • 0
  • 2461
  • 2076
  • 2537
  • 1111
  • 0
  • 1109
  • 0
  • 1056
  • 0
  • 1880
  • 1683
  • 1947
  • 2356
  • 2169
  • 1862
  • 2341
  • 588
  • 1033
  • 2195
  • 0
  • 2282
  • 0
  • 2175
  • 2508
  • 1698
  • 664
  • 1800
  • 666
  • 1560
  • 1355
  • 2193
  • 2253
  • 0
  • 1935
  • 674
  • 1977
  • 2107
  • 2527
  • 0
  • 2031
  • 1649
  • 2020
  • 2794
  • 0
  • 2247
  • 2131
  • 2389
  • 1729
  • 0
  • 2140
  • 2410
  • 2090
  • 2484
  • 2449
  • 2439
  • 2212
  • 2408
  • 1898
  • 1259
  • 2675
  • 2157
  • 0
  • 1360
  • 1555
  • 1996
  • 1409
  • 2147
  • 0
  • 2480
  • 1264
  • 0
  • 1889
  • raf
  • p44/42
  • mek
  • PLCg
  • PIP3
  • PIP2
  • jnk
  • p38
  • AKT
  • PKA
  • PKC
  • MIT Supercomputer

Clustering of Biosignature, Clinical Significance

  • Irish JM, Hovland R, Krutzik PO, Perez OD, Bruserud O, Gjertsen BT, Nolan GP. Single cell profiling of potentiated phospho-protein networks in cancer cells. Cell. 2004 Jul 23;118(2):217-28.
  • Thank You for your time


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