FLOW CYTOMETRY

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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_at_iupui.edu
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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.

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Flow Cell
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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.

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Fluorescence Activated Cell Sorting
488 nm laser
FALS Sensor
Fluorescence detector
-
Charged Plates
Single cells sorted into test tubes
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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.

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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

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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)
• Forward Angle Light Scatter 2 - 20 (FSC), size
  related biased by refractive index
• Side Scatter near 90 (SSC), structure dependent
  - reflective qualities of a particle

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Forward Angle Light Scatter
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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)
• Forward Angle Light Scatter 2 - 20 (FSC), size
  related biased by refractive index
• Side Scatter near 90 (SSC), structure dependent
  - reflective qualities of a particle

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90 Degree Light Scatter
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Types of Optical Filters
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Spectra of Fluorochromes Dyes Used on Research
Cytometers - 2 Excitation - 488nm
Argon / 535nm HeCad
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Typical 4 parameter layout

530nm band pass FL1
PMT
488nm band pass FSC
488nm laser beam
PD
flow cell
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FACScan - FACS Calibur Examples of commonly
used fluorochromes
PerCP TruRed PI 1 PE-Cy5 PE-Cy7 Red613 2
Argon Laser
FL 3
670 LP
488nm
FITC GFP FMLP CFSE DCFH BODIPY DHR 123
TOTO-1 TO-PRO-1 Fluo-3 Calcein Alexa 488
90 light scatter Granularity
PE Cy3 PI
FL 2
610 LP
585/42 BP
FL 1
560 SP
530/30 BP
1 PI is dimmer than in the FACScan (650nmLP) 2
Red613 is extremely poor with the 670LP
FSC
FACScan family 3 to 4 Fluorescence Detectors
2 - 16 light scatter Size, refractive index
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Multifaceted polygon assembly of filters and
detectors
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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

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Histograms or Bivariate Displays?
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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

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Histograms or Bivariate Displays?

• Bivariate displays often provide more graphical
  information

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Dot/Density/Contour
RK-04-17-98.004
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So what can we measure by flow cytometry?
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Light Scatter Gating
Side Scatter Projection
Neutrophils
Scale
1000
200
100
50
40
Monocytes
30
20
15
Lymphocytes
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200
400
600
800
1000
0
90 Degree Scatter
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Immunophenotyping
1000
100
10
1
.1
Log FITC
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Immunophenotyping
CD2
CD4
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Normal Cell Cycle
M
G0
G2
DNA Analysis
G1
s
Count
s
0
200
400
600
800
1000
DNA content
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DNA Analysis
0
200
400
600
800
1000
4N
2N
PI Fluorescence
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Calcium Flux - Indo-1
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Multifaceted polygon assembly of filters and
detectors
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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

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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 (lt0.05)
• 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

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T Cell Subsets are Defined by Multiple Parameters
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What other information can be collected from
immunofluorescence?
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Examples - histogram
Geometric mean/median
Arithmetic mean
Geometric mean 281.88 Arithmetic mean
455.80 Median 283.87
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Differences/Similarities between Flow and Imaging
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B
A
C
R2
R3
E
D
R2
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Differences/Similarities between Flow and Imaging
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Mean/Median - histogram example
Marker Mean Median Gmean M1 18.0
13.0 15.0 M2 976.0 583.0
626.0
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Differences/Similarities between Flow and Imaging
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Cytometer signal pathsimplified 2 color example
lens
Cell
Cytometer
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Flow Cytometry versus Flow Activated Cell
Sorting (FACS)
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