Flow Cytometry Basics:
What, How and Why?
Flow Cytometry: The 30,000 Foot View
What?
 High speed, low resolution measurement of the properties of individual
cells (or other particles) entrained in a flowing liquid (usually saline).
How?
 A flow cytometer is an instrument that detects and measures single
cells/particles in suspension as they pass through a sensing region
 Use of fluorescent probes allows different biological properties of a
cell to be quantified based on its interaction(s) with varying
wavelengths of light.
Why?
 Faster, more sensitive and more quantitative measurement of cell
properties than fluorescence microscopy
 Multicolor single cell analysis allows subpopulations present in a
mixture to be characterized without first physically isolating them
 Even very rare cells can readily be detected
 In flow cytometers with sorting capability, selected subpopulations of
cells with defined properties can be physically retrieved for study
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Flow cytometry basics: How?

Instrumentation
•
•
•
•
•

lasers
fluidics
optics
electronics
sorting
Data display and analysis
• Single parameter (“histograms”)
• Multiparameter (“dot plots”, “contour plots”)
• Listmode data and gating

Fluorescence measurements
•
•
•
•
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Excitation and emission
Fluorochromes
Filters
Color compensation (correction for color overlap)
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HOW NOT TO BE
A FLOW CYTOMETRIST
Drawing by Ben Givan
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Flow Cytometry vs. Microscopy
Low throughput, high resolution
High throughput, low resolution
DETECTORS
LASER
PMT 1
PMT 2
LIGHT
SOURCE
Adapted from figure kindly provided by Dr. Frank Mandy (Health Canada)
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Flow Cytometry:
Single Cell Analysis in a Fluid Stream
PMT
4
3
2
1
LASER
Figure courtesy of Dr. Frank Mandy (Health Canada)
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Why Use Flow Cytometry?



Can readily detect
discrete and/or rare
subpopulations
Can quantify
differences between
subpopulations
without physical
separation
Can be combined
with flow sorting, to
allow physical
isolation and
characterization of
specified subsets
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Starting Material: Stained Cells
What a Flow
Cytometer Can (and
Cannot) Tell You
Photomicrographs courtesy of
Dr. Paul Wallace (RPCI)
Cultured U937 cell stained with propidium iodide
0 Hours at 37º C.
2 Hours at 37º C.
18 Hours at 37º C.
Monocytes stained with fluorescein-tagged antibody to FcR1 (green fluorescence) and re-stained with phycoerythrintagged antibody to FcR1 (red fluorescence) after varying incubation times to allow receptor internalization
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What Does A Flow Cytometer Look Like?
(10,000 Foot View…)
Block diagram courtesy of John Martin
(Los Alamos Nat’l. Flow Cytometry Resource)
Basic ingredients
• Fluidics to deliver stream of particles single file
to the analysis point where light beam is focused
• Light source & focusing optics
• Filters, detectors & electronics to detect particlelight interaction and convert to digital format
• Computer to record and analyze digitized data
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What Does A Flow Cytometer Look Like?
(Up Close and Personal…)
“Jet in air”
(most common
configuration for
instruments that
both analyze and
sort cells)
Fluorescence
& right angle
light scatter
pickup lens
Glass flow cell
(most common
configuration for
instruments that do
high sensitivity
analysis but not
cell sorting)
Photograph courtesy
of John Martin
(Los Alamos Nat’l.
Flow Cytometry
Resource)
Forward light
scatter detector
Quartz flow cell &
Lasers
sensing region
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What Happens When a Cell Interacts With Light?
(It Depends…!)
Figure adapted from J. Treadway, (Quantum Dot Corp.)
Light is elastically scattered in all
directions (no change in color/
wavelength)
• Scatter at low angles from the
exciting light beam is a function of
cell size and refractive index
• Scatter at high angles is a function of
surface “roughness” or cytoplasmic
granularity
Interaction of exciting light and
dye(s) or naturally occurring cell
components leads to fluorescence
• Emitted light differs in color from
exciting light (longer wavelengths)
• Emitted intensity a function of
number of dyes per cell
Sources of cellular fluorescence
• Intrinsic: naturally occurring fluors – e.g. flavins, chlorophyll, other
“autofluorescent” cellular components
• Extrinsic: deliberately by the experimenter to reflect different biological or
biochemical properties – e.g. DNA binding dyes, antibodies, etc.)
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Why Use Fluorescence?
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Cell Interrogation
Quartz Flow Cell
Laser
Beam
Shaping
Lens
Figure courtesy of Dr. Paul Wallace (RPCI)
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Q: HOW Do Flow Cytometers “See” Colors?
A: Through wavelength-selective filters
530 bp
Filter
580 bp
Filter
FL1 PMT
560 SP Dichroic
Mirror
FL2 PMT
500 LP Dichroic
Mirror
SS PMT
FS
Photodiode
Laser
Quartz Flow
Cell
Figure courtesy of Dr. Paul Wallace (RPCI)
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Getting Inside the Black Box: How Are
Cell/Light Interactions Converted to Digital Data?
Block diagram courtesy of John Martin
(Los Alamos Nat’l. Flow Cytometry Resource)
Basic ingredients
• Fluidics to deliver stream of particles to analysis
point where light beam is focused
• Light source & focusing optics
• Filters, detectors & electronics to detect particlelight interaction and convert to digital format
• Computer to record and analyze digitized data
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Photodetectors: Turning Light into Electrons
Input
•Scattered laser light
•Fluorescence
(multiple colors)
Output
Electric current
(10-6 – 10-9 amps)
(Electron avalanche
catcher!)
(Emits electrons
when hit by light)
(Amplify photoelectrons;
voltage dependent)
Figures courtesy of John Martin
(Los Alamos Nat’l. Flow Cytometry Resource)
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Turning Electrons into Digital Pulses: Step 1
PMT
Condenser
Laser
Quart Flow
Cell

Peak

Area under the curve

Width or time
List Mode File
pulse height
1000
0
time
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Figure courtesy of Dr. Paul Wallace (RPCI)
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Turning Electrons into Digital Pulses: Step 2
Using hardwired circuitry
(older flow cytometers, slower electronics)
Cell Cell
Enters Exits
beam Beam
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Using digital signal processing
(newer flow cytometers, fast electronics)
Cell Cell
Enters Exits
beam Beam
Figures courtesy of John Martin
(Los Alamos Nat’l. Flow Cytometry Resource)
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Getting Inside the Black Box…
What Does the Data Look Like?
Block diagram courtesy of John Martin
(Los Alamos Nat’l. Flow Cytometry Resource)
Basic ingredients
• Fluidics to deliver stream of particles single file
to the analysis point where light beam is focused
• Light source & focusing optics
• Filters, detectors & electronics to detect particlelight interaction and convert to digital format
• Computer to record and analyze digitized data
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Raw FCS Data File (untranslated)
$F
Figure courtesy of A. Givan
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FCS Data file (translated)
Parameter value
(channel #, proportional to signal intensity)
Event #
Figure courtesy of A. Givan
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Do It Yourself Cytometry
(in-class exercise)
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LASER
PMT 1
Number of events
Single Parameter Data Display: Frequency Histogram
1
256
512
768
Channel Number
[low]
[medium]
1024
[high]
[Relative Fluorescence Intensity]
Adapted from figure kindly provided by Dr. Frank Mandy (Health Canada)
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Reporting the Data: Single Parameter Histograms
Measures of peak location/signal
intensity
1) Modal Channel Number (most
commonly observed value)
2) Median Channel Number (equal #
of events above and below)
3) Mean Channel Number (average
value; for Mean Fluorescence
Intensity often abbreviated MFI)
Measures of peak width/signal
heterogeneity
1) Standard deviation (SD)
2) Coefficient of Variation (CV)
= relative standard deviation
= SD/mean x 100
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Mode: a = b; a, b > c
Median: a = b; a, b < c
Mean: a = b = c
SD, CV: c >> a > b
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Single Color Immunofluorescence Analysis
M1 = analysis Marker ,
region 1 for FL1
( dim – bright FL1)
M1 = analysis Marker,
region 1 for FL2
( dim – bright FL2)
(Raw channel numbers using 4 decade log amps)
% Positive
48 %
% Positive
12 %
Adapted from figure kindly provided by A. Givan
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++
A
+
A
--
+
B
A
Two Parameter Data : 1+1 = 4 (not 2)
B
B
NOTE: ++ events could be both colors on one cell OR 2 single color cells stuck together
Figure courtesy of Dr. Frank Mandy (Health Canada)
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Two-color data display
(dot plot) and analysis
(quadrant statistics)
Note logarithmic scale
display showing relative
fluorescence intensities (not
raw channel numbers)
Gated Events: 2823
Quad
UL
UR
LL
LR
Events % Gated X Geo Mean Y Geo Mean
295
10.45
2.37
243.05
6
0.21
97.05
51.40
276
9.78
4.37
3.60
2246
79.56
212.64
1.78
Figure courtesy of A. Givan
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“Dot” Plot
Signal Intensity 
Different Types of Two-Parameter Data Plots
“Density”
Plot
Signal Intensity 
“3D” Plot
“Contour”
Plot
Figure courtesy of A. Givan
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Two Kinds of Light Scatter: Leukocyte Differential?
Basophils
Side Light Scatter (SS)
Granulocytes
Neutrophils
Eosiniphils
Monocytes
Lymphocytes
Forward Light Scatter (FS)
Mid 1980’s
Figure courtesy of Dr. Frank Mandy (Health Canada)
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Light Scattering Properties Reflect Intrinsic
Differences Among Cells (and Viewers)
FSC = forward or “low angle” scatter; strongly influenced by cell size
SSC = side or “right angle” scatter; reflects granularity or internal structure
Monocytes
RBCs, debris
Lymphocytes
RBCs, debris
Lymphocytes
Signal Intensity 
Granulocytes
Granulocytes
Monocytes
Signal Intensity 
“Beckman Coulter” display
“Becton Dickinson” display
PLOT IT ANY WAY YOU LIKE!
Figure courtesy of A. Givan
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Light Scatter: It’s All Relative
(Same sample, two different amplifier settings)
Figure courtesy of A. Givan
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Electronic “Gating”: The Power Behind Flow Cytometry
MONOS
PMNS
LYMPHS
Figure courtesy of A. Givan
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Light Scatter Gating In Action
2P Dot Plots
Gated Data
413
629
862
3
965 662
322
660
4
470 204
540
210
5
692 997
280
373
6
925 501
563
838
7
410 950
506
841
8
409 286
128
178
9
344 790
193
15
629
862
540
210
128
800
231
432 184
600
771 958
2
G1 gate values:
178
G1
FS:
300-610
SS:
50-400
0
1
CD3 FITC CD4 PE
400
SC CD3 FITC CD4 PE
Side Scatter
FS
200
Raw Data
Event
1000
Listmode Data File (translated)
0
700 820
244
301
11
643 898
232
114
12
757 373
327
649
13
525 175
582
201
582
201
14
512 164
511
857
511
857
15
383 149
584
157
584
157
16
798 353
290
654
17
451 168
596
780
596
780
18
385 174
574
59
574
59
19
434 164
551
130
551
130
20
1023 881
574
428
21
730 111
101
163
22
424 738
586
91
23
699 893
351
277
24
645 950
245
279
10,000 597 163
568
862
0
200
400
600
800
1000
102
100
101
CD4 PE
103
104
Forward Scatter
100
101
102
103
104
CD3 FITC
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Figure courtesy of Dr. Paul Wallace (RPCI)
Inc.
Flow Essentials SciGro,
04.04.17 (p.
33)
Light Scatter Gating: Differentiating Among Leukocytes
(no physical separation required)
MONOS
PMNS
LYMPHS
Figure courtesy of A. Givan
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Fluorochromes for Antibody Tagging:
Choice, choices….
Probe
FITC
PE (Phycobiliprotein)
APC (Phycobiliprotein)
PerCP™ (Phycobiliprotein)
Cascade Blue
Coumarin
Texas Red™
Tetramethylrhodamine
CY3 (indotrimethinecyanines)
CY5 (indopentamethinecyanines)
Excitation
488
488
630
488
360
350
610
550
540
640
Emission
525
575
650
680
450
450
630
575
575
670
Courtesy of J. Paul Robinson, Purdue Univ. Cytometry Facility
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Useful References and Resources
FLOW CYTOMETRY
 Melamed, M.R., Lindmo, T. and Mendelsohn, M.L., Eds., Flow Cytometry and
Sorting, Second Edition, Wiley-Liss (1990).
 Darzynkiewicz, Z., Robinson, J.P. and Crissman, H.A., Eds., Flow Cytometry,
Parts A and B (Methods in Cell Biology, Volumes 41 and 42), Academic Press
(1994).
 Shapiro, H.S. Practical Flow Cytometry (4th Ed.), Wiley-Liss (2003);
read-only version available online at
http://probes.invitrogen.com/products/flowcytometry/practicalflowcytometry.html.
 Robinson, J.P., Ed., Current Protocols in Cytometry, John Wiley and Sons (1997).
 Givan, A.L., Flow Cytometry: First Principles (2nd Ed), Wiley-Liss (2001).
 http://www.isac-net.org/ (International Society for Advancement of Cytometry
home page; especially links for “Presentations/Lectures”, “Purdue Discussion” and
“Jobs/Positions”
 http://www.cytometry.org/ (Clinical Cytometry Society home page)
 http://wiki.clinicalflow.com/ (Clinical flow wiki, sponsored by DeNovo Software)
 http://flowbook-wiki.denovosoftware.com/ (wiki version of “Flow Cytometry –
A Basic Introduction” by M.G. Ormerod, sponsored by DeNovo Software)
 http://www.vsh.com/cytometrycourses/ (listing of applications-focused workshops
and courses, sponsored by Verity Software House)
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