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Flow Cytometry - Graduate progra

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									       FLOW CYTOMETRY

            EDWARD F. SROUR, Ph.D.
Professor of Medicine, Pediatrics, & Micro/Immunol
      Indiana University School of Medicine
                 Indianapolis, IN
                esrour@iupui.edu
  What is Flow Cytometry?
• Cytometry refers to the measurement of physical/chemical characteristics of
cells or other biological particles.

• Flow Cytometry is the process whereby such measurements are made upon
cells/particles as they pass through a measuring apparatus (hopefully in single
file) suspended in a fluid stream.

• Flow Sorting (Flow Cytometric Cell Sorting) extends flow cytometry with
the additional capacity to divert and collect cells exhibiting an identifiable set of
characteristics either mechanically or by electrical means (Flow Cytometric
Analysis).

• FACS - Fluorescence Activated Cell Sorting? FACS is a trademark of
Becton Dickinson Immunocytometry Systems (BDIS). All FACS instruments
are BDIS systems, but not all cytometers are FACS.
                  Flow Cell

                       Injector
                          Tip     Sheath
                                   fluid




Fluorescence
       signals

  Focused laser
         beam
  What is Flow Cytometry?
• Cytometry refers to the measurement of physical/chemical characteristics of
cells or other biological particles.

• Flow Cytometry is the process whereby such measurements are made upon
cells/particles as they pass through a measuring apparatus (hopefully in single
file) suspended in a fluid stream.

• Flow Sorting (Flow Cytometric Cell Sorting) extends flow cytometry with
the additional capacity to divert and collect cells exhibiting an identifiable set of
characteristics either mechanically or by electrical means (Flow Cytometric
Analysis).

• FACS - Fluorescence Activated Cell Sorting? FACS is a trademark of
Becton Dickinson Immunocytometry Systems (BDIS). All FACS instruments
are BDIS systems, but not all cytometers are FACS.
Fluorescence Activated
Cell Sorting

                      488 nm laser           FALS Sensor


                                     Fluorescence detector



          Charged Plates     -        +


     Single cells sorted
     into test tubes
  What is Flow Cytometry?
• Cytometry refers to the measurement of physical/chemical characteristics of
cells or other biological particles.

• Flow Cytometry is the process whereby such measurements are made upon
cells/particles as they pass through a measuring apparatus (hopefully in single
file) suspended in a fluid stream.

• Flow Sorting (Flow Cytometric Cell Sorting) extends flow cytometry with
the additional capacity to divert and collect cells exhibiting an identifiable set of
characteristics either mechanically or by electrical means (Flow Cytometric
Analysis).

• FACS - Fluorescence Activated Cell Sorting? FACS is a trademark of
Becton Dickinson Immunocytometry Systems (BDIS). All FACS instruments
are BDIS systems, but not all cytometers are FACS.
Measurements in Flow Cytometry

• Electronic Cell Volume
   – detect and measure the volume of particles as they pass through a
     small orifice
   – viable cells are better insulators than fixed/dead cells
• Light Scatter
   – all objects passing through a laser beam in a cytometer will scatter
     light
   – large objects will scatter more light in the forward direction than
     small objects
   – light scatter signals are commonly used to trigger data acquisition
Measurements in Flow Cytometry

• Light Scatter (continued)
   – Two angular regions for detecting light scatter (usually from the
     primary laser only in a multi-laser system)
       1 Forward Angle Light Scatter 2° - 20° (FSC), size related biased by
         refractive index
       2 Side Scatter near 90° (SSC), structure dependent - “reflective”
         qualities of a particle
        Forward Angle Light Scatter



Laser

                                  FALS Sensor
Measurements in Flow Cytometry

• Light Scatter (continued)
   – Two angular regions for detecting light scatter (usually from the
     primary laser only in a multi-laser system)
       1 Forward Angle Light Scatter 2° - 20° (FSC), size related biased by
         refractive index
       2 Side Scatter near 90° (SSC), structure dependent - “reflective”
         qualities of a particle
        90 Degree Light Scatter


Laser

                                  FALS Sensor




                  90LS Sensor
                    Types of Optical Filters
         Wavelengths

             LONG (700nm)
                             Short Pass        Long Pass         Band Pass

Pass Through
   Filters
                             650 Short Pass    550 Long Pass   600/100 Band Pass
             SHORT (500nm)      (600SP)           (650LP)           (600/100)

          Transmitted           <650 nm          >550 nm          550 - 650 nm
                                                                    (600±50)
              Blocked           >650 nm          <550 nm       <550 nm & >650 nm

              LONG (700nm)



  Dichroic
   Filters
                              650 Short Pass   550 Long Pass
             SHORT (500nm)       (600SP)          (650LP)

             Transmitted        <650 nm           >550 nm
             Diflected 90°      >650 nm           <550 nm
Spectra of Fluorochromes Dyes Used on Research Cytometers - 2
              Excitation - 488nm [Argon] / 535nm [HeCad]
  Typical 4 parameter layout
                           PMT        530nm band pass
                                      FL1

                                            560nm short pass
585nm band pass   PMT
                                            dichroic mirror
FL2


488nm band pass   PMT                       510nm long pass
SSC                                         dichroic mirror




                                             488nm band pass FSC
       488nm laser beam                PD
                          flow cell
                 PerCP                                     FACScan - FACS Calibur
                 TruRed
                                                   Examples of commonly used fluorochromes
                 PI   1


                 PE-Cy5
Argon                                 FL 3
                 PE-Cy7                                             APC
Laser
                 Red613 2                     670 LP                Cy5
488nm
                                                                    TOTO-3                                 FITC
                      Half Mirror                            FL 4               90° light scatter
                                                                    TO-PRO-3                               GFP
                                               661/16 BP                        “Granularity”
                                                                                                           FMLP
                                                                    PE
633nm                     610 LP                             FL 2                                          CFSE
                                                                    Cy3        SSC
                                                                                                           DCFH
                                               585/42 BP            PI
                                                                                                           BODIPY
                                                                                                    FL 1   DHR 123
                                     560 SP
                                                                                     530/30 BP             TOTO-1
                          1   PI is dimmer than in the FACScan (650nmLP)
                          2   Red613 is extremely poor with the 670LP                                      TO-PRO-1
           FSC                                                                                             Fluo-3
                                               FACScan family
                                                                                                           Calcein
2° - 16° light scatter                 3 to 4 Fluorescence Detectors
                                                                                                           Alexa 488
“Size”, refractive index
Multifaceted polygon assembly of filters and detectors
 The most common display formats
• Histogram
   – single parameter only, array created
   – acquisition and analysis
• Dot Plot
   – bivariate, two parameters (scattergram)
   – acquisition and analysis
• Density Plot
   – bivariate, 64x64, 128x128, or 256x256 2D array
   – acquisition and analysis
• Contour Plot
   – bivariate, 64x64, 128x128, or 256x256 2D array
   – analysis only
Histograms or Bivariate Displays?
 The most common display formats
• Histogram
   – single parameter only, array created
   – acquisition and analysis
• Dot Plot
   – bivariate, two parameters (scattergram)
   – acquisition and analysis
• Density Plot
   – bivariate, 64x64, 128x128, or 256x256 2D array
   – acquisition and analysis
• Contour Plot
   – bivariate, 64x64, 128x128, or 256x256 2D array
   – analysis only
    Histograms or Bivariate Displays?
•   Bivariate displays often provide more graphical information


                                                        RK-04-17-98.004
                    Dot/Density/Contour
                 RK-04-17-98.004




Dot Plot      Density Plot            Contour Plot
acquisition   showing distributions   showing populations
rare events   relative numbers of     relative numbers of
              events                  events
So what can we measure by flow cytometry?
                         Light Scatter Gating
                                               Side Scatter Projection




                          1000
                                               Neutrophils




                                                                             Forward Scatter Projection
Scale
                          800
Forward Scatter




                  1000
                  200
                  100
                          600




                  50
                  40
                  30                                     Monocytes
                          400




                  20
                  15
                                                   Lymphocytes
                          200




                  8
                                 0




                                     0   200       400    600   800   1000

                                                  90 Degree Scatter
                     Immunophenotyping
   5
   1




            0
  1
  1




            0
        7
Count


            0
        3



            0
            0




                .1      1     10       100   1000

                            Log FITC
            Immunophenotyping


                                    CD2
            CD4

CD # = cluster designation number
         Normal Cell Cycle
     M                G0
G2                                DNA Analysis
         G1
                                  G0G1
s
              C
              o
              u
              n
              t
                                        s         G2 M
                  0        200    400       600    800   1000
                                 2N          4N
                                   DNA content
         300
         225
         150                  DNA Analysis
Counts

           75
                0




                    0   200         400   600        800   1000

                               2N               4N
                                PI Fluorescence
                     Calcium Flux - Indo-1


                        1000
                        800
RATIO [short/long]




                        600
                        400
                        200




                                                 Stimulation
                               0




                                   0   36   72    108        144   180
                                            Time (Seconds)
Multifaceted polygon assembly of filters and detectors
Uses for Multiple Fluorescence Parameters

• Phenotyping (Cell Surface Antigens)
   – 168 CD Cell Surface Antigens
   – Many functional populations require 5 or more surface
     markers to be fully distinguished
• Functional Assays
   – Cell Cycle (PI, BrdU, Intracellular Cyclins)
   – Apoptosis (AnexinV, Active Caspase-3)
   – Ca++ Flux [Indo-1, Fura]
• Cytokine Production
• Intracellular Signaling (Rb phosphorylation)
• Gene Reporter [Molecular] Assays
   – GFP, BFP, YFP, NFP Expression
   – LacZ Expression
Advantages/ Disadvantages of Using More Colors

 • Advantages
    – Save Time, Reagents, Samples-
       • (1) 6-color stain = (15) 2-color stains
    – Exponential increase in information
       • Data from (1) 6-color stain » (15) 2-color stains
    – identify new/rare population (<0.05%)
    – internal controls
 • Problems
    – Must carefully choose combinations of
      fluorochrome conjugates
       • All reagents not available in all colors
    – Greater potential for errors in compensation
       • Proper controls required
T Cell Subsets are Defined by Multiple Parameters

                  Lymphocytes in Blood
      B cells                                            N K cells
                               T cells
   CD5+     CD5–                                              CD 16+CD 56–
      CD 23+                          CD 4–CD 8–              CD 16+CD 56+
      CD 23–                                                  CD 16–CD 56+
         CD 45RB+
         CD 45RB–
                                                            CD8 dim
          TH (CD4+)           TC/S (CD 8+)                       CD 4+
                  N aive               N aive                    CD 4–
   Memory                    Memory

       CD 11a+    CD 60+          CD 11a+       CD 60+
                  CD 60–                        CD 60–
                                                             T cells
       CD 11a–    CD 60+          CD 11a–       CD 60+                   V9+
                  CD 60–                        CD 60–            V2
                                                                         V9–
       CD 45RA+                   CD 45RA +                       V1
                    CD 57+                       CD 57+
                    CD 57–                       CD 57–
What other information can be collected
      from immunofluorescence?
      Examples - histogram
Geometric mean/median


 Arithmetic mean


                        Geometric mean: 281.88
                        Arithmetic mean: 455.80
                        Median:          283.87
   Differences/Similarities between Flow and Imaging

                    Property                        Flow         Imaging
Population analysis/Statistics                        +             -
Single cell details/architecture                      -             +
Live cell sorting (analysis??)                         +              -
Analysis in native microenvironment                    -              +
Analysis of cellular functions (e.g. Ca++ influx)      +               -
Need to use a substrate for cell “support”              -             +
                                                    Relatively
Multiple parameter analysis (>5)                      easy
                                                                 More difficult

Simultaneous analysis of multiple parameters          +/-            +/-
Analysis of properties of a single parameter           +              +
Costly technique                                       +              +
Archiving of permanent records                         +             +/-
           Sp eci men _00 1_1 2.fcs                         Sp eci men _00 1_1 2.fcs                         Spe ci men_ 001 _12 .fcs


                                           A                                                B                                           C
                                    R2
                                    R2



                                                           R3
                                                           R3




100         101         102     103            104   100        101      102     103            104    100    101       102      103        104
                    CD3 4 APC-A                                   CD1 84 APC-Cy7-A                                  CD1 6 FITC-A
           Sp eci men_ 001 _13 _00 1.fcs
                                                            Sp eci men _001 _13 _00 1.fcs



                                           D                                                E


       R2
      R3




 100          101          102       103       104   100        101       102         103        104
                    CD1 84 APC-Cy7-A                                  CD1 27 PE-A
Tau   DAPI




             IUS
   Differences/Similarities between Flow and Imaging

                    Property                        Flow         Imaging
Population analysis/Statistics                        +             -
Single cell details/architecture                      -             +
Live cell sorting (analysis??)                         +              -
Analysis in native microenvironment                    -              +
Analysis of cellular functions (e.g. Ca++ influx)      +               -
Need to use a substrate for cell “support”              -             +
                                                    Relatively
Multiple parameter analysis (>5)                      easy
                                                                 More difficult

Simultaneous analysis of multiple parameters          +/-            +/-
Analysis of properties of a single parameter           +              +
Costly technique                                       +              +
Archiving of permanent records                         +             +/-
Mean/Median - histogram example


               Marker Mean Median Gmean
               M1    18.0    13.0    15.0
               M2   976.0   583.0   626.0
   Differences/Similarities between Flow and Imaging

                    Property                        Flow         Imaging
Population analysis/Statistics                        +             -
Single cell details/architecture                      -             +
Live cell sorting (analysis??)                         +              -
Analysis in native microenvironment                    -              +
Analysis of cellular functions (e.g. Ca++ influx)      +               -
Need to use a substrate for cell “support”              -             +
                                                    Relatively
Multiple parameter analysis (>5)                      easy
                                                                 More difficult

Simultaneous analysis of multiple parameters          +/-            +/-
Analysis of properties of a single parameter           +              +
Costly technique                                       +              +
Archiving of permanent records                         +             +/-
Cytometer signal path
simplified 2 color example
                                 diff amps   log amps


                PMT’s                                      amplified
                                                            signals
         lens           pulses
                                    linear amps



  Cell                   PD
                                             signal processing



                              computer
    Cytometer
                                  sort module
     Flow Cytometry

          versus


Flow Activated Cell Sorting
         (FACS)

								
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