Kumc introduction to flow cytometry

Introduction to Flow
Cytometry
Rich Hastings
KUMC Flow Cytometry Core Lab
3901 Rainbow Boulevard
Kansas City, KS 66160
913-588-0627
rhastings@kumc.edu
http://www.kumc.edu/flow/

Flow Cytometry
Flow (noun) = the motion characteristics of fluids.
Cytometry (noun) = is a general name for a group of
biological methods used to measure various
parameters of cells. Parameters which can be
measured by cytometric methods are cell size, the
stage of the cell cycle, the DNA content of the cell,
the existence or absence of specific proteins on the
cell surface or in the cytoplasm, to name but a few.
(Wikipedia)

Flow Cytometry
All forms of Cytometry depend on the
basic laws of physics, including those
of fluidics, optics, and electronics.
Watson, J. V. Cytometry, 38 2-14, 1999.

Flow Cytometry
Flow cytometry is a system for sensing
individual cells in a physiologic saline
solution as they move in a focused liquid
stream through a fixed laser beam scattering
light and emitting fluorescence that is
measured and converted into digitized data.

The History of Flow Cytometry
1930’s Moldavan in Science described the counting of blood cells filing through
a capillary tube using a photoelectric sensor.
1940’s US Army constructed a device that detected bacteria in a stream of air, using a Ford
headlight as a light source and a photomultiplier tube as a detector. 60% ~ 0.6 μm
1950’s Caspersson measured nucleic acid and protein metabolism for normal and
abnormal cell growth using a Cadmium spark as a UV light source.
Coulter constructed a cell counter based on the fact that the electrical conductivity
of cells is lower than that of saline. Saline conducts, cells impede.
1960’s Kamentsky at IBM developed the Rapid Cell Spectrophotometer. It used an arc lamp
source and measured nucleic acid content and cell size. A computer (!!!!) measured
and analyzed the data. IBM loaned Herzenberg a prototype and he developed the
first real FACS.
1970’s Industry takes over technical development: BD, Beckman, Ortho etc.

Frequency distribution of DNA content
Cells from a normal cervix
Cells from a cervical carcinoma
Premalignant cells from the epithelium
Quantitative cytochemical studies on normal,
malignant, premalignant and atypical cell
populations from the human uterine cervix, Acta
Cytologica 8, 1964
O. Caspersson 1964
Images from Dr. Louis Kamentsky

The Coulter Counter: Counts cells and measures
their size based.
Patent
The first commercial version of the Coulter Counter

LA Kamentsky, MR Melamed & H. Derman, Spectrophotometer:
New instrument for ultrarapid cell analysis, Science 150, 1965

Herzenberg Lab at Stanford, Early sorters and analyzers.

Flow cytometry has benefited from the
technological development of:
Monoclonal antibodies
Fluorochromes
DNA, RNA and Functional stains
Computers and the miniaturization of Electronics
Lasers

How Flow Cytometers Work
The principal of hydrodynamic focusing confines cells
to the core (sample) stream by a cell-free sheath fluid.
Injector
Tip
Sheath
fluid
(PBS)
Laser interrogates
Stream
Objective is to have one
cell pass through the
laser intercept at a time.
What makes flow cytometry
so powerful is the ability to
gather data on each cell
individually but having the
capability to analyze 1000’s
of cells/sec.

Sheath pressure is constant.
Sample pressure is variable.
Aria LSR II
Flow is laminar.
Flow cell shape allows for
hydrodynamic focusing.
Hydrodynamic focusing
causes cells to line up
along their long axes.

Higher Flow Rate = Good for Qualitative Measurements
Lower Flow Rate = Greater Resolution and Quantitative Measurements
From BD LSR II manual

Laser is an acronym for
Light Amplification by Stimulated Emission
of Radiation
1. Lasers provide light of a specific wavelength. Most lasers used
in flow cytometry operate in the visible spectrum.
2. An important aspect of laser light is coherence. Coherent light
has the same wavelength, phase and direction.
3. Modern lasers generate light that is reliable and constant.

Electromagnetic Spectrum
Violet Laser Blue Laser Red Laser
From: http://www.antonine-education.co.uk

Laser interrogation of a cell tells us physical properties of that cell.
Laser
interrogates
Cell in Flow Cell
Incident light scattered
at small angles (0.5-
2.0º) is called Forward
Scatter (FSC)
Incident light scattered
at an angle of 90º is
called Side Scatter
(SSC)

Forward Scatter (FSC)
Rough measure of size, influenced by the
wavelength of light, and the angle, lenses and
apertures that light is collected at and with.
Different flow cytometers will give slightly
different FSC measurements.
Most flow cytometers measure FSC with a
photodiode. Bacteria – Photomultiplier tube
(PMT)
Dead cells may have lower FSC measurements
than live cells. Osmotic swelling can increase cell
volume, and decrease light scatter.
FACSCalibur
Epics XL
Data: Shapiro and Becker

Forward Scatter (FSC)
“A big problem in the published literature is the use of forward scatter as a
trigger/discriminator. Whilst fairly robust for leukocyte detection it is the
most variable signal between systems and it is most alignment critical. It is
affected by refractive index mismatches between sheath and sample, beam
geometry, polarization, beam stop position, and collection angle. In some
cases the relative forward scatter position of particles of different sizes does not
follow their relative order in physical size. In jet-in-air sorters the beam geometry
and the jet undulation at the intercept are critical factors whereas in cuvette-based
instruments these tend to be dirt on the optical surfaces and slight rotation
of the flow cell to the beam axis.”
Gerhard Nebe-von-Caron
Cytometry Part A 75A: 8689, 2009

Side Scatter (SSC) is the measure of light
scattered at an angle of 90º (orthogonal).
SSC is a measure of the complexity of the cell’s internal structures.
The more ‘granular’ a cell is the higher its SSC will be.
A neutrophil is much more granular than a lymphocyte.
Measured using a photomultiplier tube.

FSC vs. SSC alone tells us a great deal about our cells.
Lymphocyte
Eosinophil
Basophil
Neutrophil
Monocyte

Pulse Processing
From BD LSRII Manual
Laser

Parameters Measured
Height is the maximum digitized intensity measured for the pulse.
Area is the sum of all pulse heights.
Width is Area ÷ Height x 64,000.
From BD LSRII Manual

Flow cytometry takes advantage of molecules fluorescing after
excitation by laser light. Fluorochromes can be conjugated to
monoclonal antibodies. Many functional stains exist that fluoresce.
Energy acquired by the absorbance of light of certain wavelengths in
these molecules drive electrons to a higher energy state in an
unoccupied orbital.
The return of the electron to ground state results in the emission of
photons of longer wavelength (lower energy).

Fluorochromes absorb energy (light) and their electrons go from a ground state
to an excited state. The electrons return to ground state by emitting light of
lower energy, therefore longer wavelength.
Figures from Flow Cytometry - A Basic Introduction
by Michael G. Ormerod

Tandem Dyes – One molecule is excited by laser light and donates the energy
to the acceptor molecule.
Figure from Jane Limer BD Application Scientist

Fluorochromes
Synthetic, Organic Dyes:
FITC, the Dylights (Thermo Fisher Scientific), Cy dyes, Alexa Fluors (Molecular Probes),
Horizon dyes (Becton Dickinson), eFluor dyes (eBioscience), Pacific Blue, Krome Orange
(Beckman Coulter), Brilliant Violet (Biolegend)
Proteins:
R-phycoerythrin (PE) is a photosynthetic pigment found in red algae. It is a 240 kDa protein
with 23 phycoerythrobilin chromophores per molecule. Very bright, and excellent as the
donator molecule in a tandem fluorophore.
Allophycocyanin (APC) is a photosynthetic pigment found in bluegreen algae. APC is 105 kDa
and has six phycocyanobilin chromophores per molecule. Very bright, and excellent as the
donator molecule in a tandem fluorophore.
Green Fluorescent Protein is “genetically encoded fluorescence” encoded by a single gene. A
whole family of fluorescent proteins has originated from GFP.

The photons emitted by excited fluorophores are routed to Photo-
Multiplier Tubes (PMT).
1. Voltage is applied to the PMT making electrons present for the
absorption of light energy from photons.
2. As more photons are detected, more electrons are recruited
yielding a greater current on the detector.
3. IMPORTANT! If the PMT voltage is increased the same number of
absorbed photons will have a greater current output, increasing the
sensitivity of the PMT.

The fluorescence intensity measured is proportional to the
number of fluorescent molecules bound to the cell.
From Applied Cytometry

The Stokes Shift is the difference between the emission and
excitation wavelength.

Flow cytometers are engineered with precise light pathways.
Light is routed through three different types of Filters.

Cell
Forward light scatter 0º
A measure of cell size.
488 nm
Side scatter 90º
A measure of cell granularity.

488 nm Light path
in the LSRII

405 nm Violet Laser
Bandpass Filter Fluorochromes
440/40 nm DAPI, Horizon V450, Alexa
405, Pacific Blue, Brilliant
Violet 421
525/50 nm Pacific Orange, Cascade
Yellow, Horizon V500,
Brilliant Violet 570

488 nm Blue Laser
780/60 nm PE-Cy7
695/40 nm PE/Cy5, PE/Cy5.5, PerCP,
PerCP-Cy5.5
610/20 nm PI, PE-Texas Red
575/26 nm PE, Cy3
530/30 nm GFP, FITC, Alexa Fluor 488,
CFSE

633 nm Red Laser
780/60 nm APC/Cy7
710/50 nm Alexa Fluor 700
660/20 nm APC, Cy5, Alexa Fluor 647

Visualizing Flow Cytometry Data
Dot plot:
One parameter vs.
another.
Contour plot:
One parameter vs.
another showing
the probability
contouring.
Density plot:
One parameter vs.
another, very good
for viewing the
frequency of
subpopulations.
Histogram plot:
One parameter
only. Y-axis is the
count. X-axis is
fluorescence
intensity.

Visualizing Flow Cytometry Data
Flow cytometry data: Iterative, Derivative, Visual, as well as Statistically Powerful.

Gating
Gate on your Cells of Interest,
The Population Hierarchy is your Friend.

Gating
Gate on your Cells of Interest,
Where are your Dead Cells?

Fluorescence Minus One (FMO)
Unstained and Isotype Controls vs. FMO
PE FMO
Absence and Presence of CD4 PE
Improper Compensation
From FlowJo website/Mario Roederer

Doublets
Doublet
Discrimination:
Two cells passing
through the laser
intercept
concurrently.
Doublet
Discrimination:
Doublets have
double the area and
width values of
single cells.

Doublet Discrimination – Cell Cycle
Voltage Intensity
G0
G0
G0
G2/M
2N Height 2N + 2N Height
4N Height
2N Width 2N + 2N Width
Stained nuclei
separated by
cytoplasm
4N Width
Time

Doublet Discrimination – Cell Cycle
Blue cells are the
G0/G1 doublets.
They have double
the area and width
values of single
cells but lower
height values than
the G2/M cells.

Doublet Discrimination – Whole
Cells
A Good Tool for Gating:
Doublets have Double the Width Value while maintaining Same Height Value.
Blue Cells are singlets,
Red Cells are doublets.

Compensation
Compensation/Spectral Overlap
Most fluorochromes and dyes excited by laser light have long
emission curves.
Flow cytometers have filter sets optimized for specific
wavelengths of light.
Unfortunately, overlapping emission wavelengths from one
fluorochrome may spillover into the filter of another.

Compensation
Visualizing Spectral Overlap

Compensation
Compensation Controls
 Compensation is very important. If one fluorochrome leaks into another’s
channel, your data cannot be interpreted properly.
 Each experiment needs an unstained control and each fluorochrome
singularly.
As long as your run the proper controls, we can determine the correct
compensation values post acquisition.
 Beads can be substituted for cells if cell number is a limiting factor.
Spherotech, Invitrogen and Becton Dickinson offer anti-Ig beads that will
bind fluorescently-labeled antibodies.

Compensation
The FITC signal is leaking into the PE detector.
We can adjust the PE-%FITC spectral
overlap value until the unstained and FITC have
the same mean value for PE.

Compensation
This figure is the Diva Compensation layout for a
seven color experiment.
FACS DIVA has a module designed for
computing compensation.
Module requires an unstained control and
each color individually.
Choose colors, then gate on the cells by FSC vs.
SSC, and follow the layout.

Compensation (Pattern Recognition)
Uncompensated Data
Compensated Data

Compensation
Statistics for a Four Color Experiment
Mean Fluorescence Intensity Values

Compensation
Uncompensated Data
Voltages
PE = 538
PerCP = 791
APC = 690
Compensated Data
Comp Matrix
PerCP -%PE = 22.01
APC - %PE = 0.09
APC - %PerCP = 4.54
PerCP - %APC = 1.04

Compensation
Uncompensated Data
Voltages
PE = 650
PerCP = 825
APC = 725
Compensated Data
Comp Matrix
PerCP -%PE = 7.06
APC - %PE = 0.04
APC - %PerCP = 4.67
PerCP - %APC = 0.96

FlowJo Compensation
FlowJo is a third party
analysis software from
Treestar, Inc.
FlowJo has a built in
Compensation
Matrix/Wizard that is
powerful and intuitive.
Need single color and
unstained controls.

Compensation
Annexin-FITC and Propidium iodide (PI) need to be compensated. This
can be difficult depending on where the cells are in the stages of apoptosis.
We recommend Annexin-APC and PI, less spectral overlap.

Flow Cytometry Statistics
For Histograms:
y-axis = Number of cells/channel
x-axis = Fluorescence intensity of
designated parameter
For Dot Plots:
y-axis and x-axis = Fluorescence
intensity of designated parameters
Flow Cytometry is a qualitative assay, flow results depict the characteristics of
your sample. Samples are measured in a dimensionless unit termed
Fluorescence Intensity.

Flow Cytometry Statistics (Diva)
• Number of events - total number of events in
the defined population.
• Parent - name of the next population up in the
hierarchy.
• %Parent - number of events in the defined
population divided by the number of events in
the parent gate (next population up in the
hierarchy), expressed as a percentage.
• %Grandparent - number of events in the
defined population divided by the number of
events in the grandparent gate (two populations
up in the hierarchy), expressed as a percentage.
• %Total - number of events in the defined
population divided by the total number of events
in the tube (all events), expressed as a
percentage.

Mean - Average linear value for events in the defined population, defined as:
where n = number of events in the population, and Xi is a
value for a particular parameter, where i = 1 to n.
Geometric mean - Logarithmic average of the events in the defined
population. This mean is less sensitive to outliers than the regular mean. The
geometric mean is defined as:
where n = number of events in the population, and Xi is a
value for a particular parameter, where i = 1 to n.

Two measures are generally made of a distribution: intensity and spread.
In flow cytometry, the intensity of a distribution can be represented by the
position of the “center” of the distribution. The “center” is usually represented
mathematically by the mean, median or peak channel number.
If the data has been displayed on a linear scale, the arithmetic mean is used;
for logarithmically displayed data, the geometric mean is generally chosen.
If any part of the distribution lies off scale at either end of the axis, the value for
the mean channel number will be inaccurate and should not be used; the
median channel can be used as long as more than half of the distribution in on
scale.
Flow Cytometry - A Basic Introduction
Michael G Ormerod

The peak channel number is an inaccurate measure of the center of a
distribution and is not recommended.
For a Guassian (normal) distribution, these three values should be equal.
The spread of a distribution is usually expressed as
the Standard Deviation (SD). However, in flow cytometry, the coefficient of
variation (CV) is preferred because it is dimensionless and, on a linear scale,
does not depend on where in the histogram the data is recorded.(CV =
SD/mean channel number).
Flow Cytometry - A Basic Introduction
Michael G Ormerod

Immunophenotyping
Cell Staining Overview
 Stain 105-106 cells/tube
 Tubes vs. Plates
 Stain cells in small
volumes.
 Titer antibodies.
 Block Fc with species
specific antibodies.
 Direct vs. Indirect staining
 Fixation

Immunophenotyping-Antibody
Dilution
Most antibody manufacturers advise a dilution to start with.
We advise performing a dilution curve.
Figure from Flow Cytometry - A Basic Introduction
by Michael G. Ormerod

Immunophenotyping-Dilution
Protocol
43.3%, Mean = 6982
37.3%, Mean = 4923
36.6%, Mean = 4958
32.4%, Mean = 3784
32.4%, Mean = 3886
2.14%, Mean = 1042
1.4%, Mean = 886
32.4%, Mean = 3886
2.14%, Mean = 1042
1.4%, Mean = 886

Immunophenotyping
Gate on physical
properties of
cells.
Then, gate on the
live cells. 7AAD
negative cells.
Then, gate on
CD3+, CD56-
T-cells.
Then, gate on
CD4+ vs. CD8+.

Immunophenotyping
Multi-color experiments – Overcome the Complexity

Setting up an Experiment
From Lora Barsky, USC Flow Core

Immunophenotyping
Multi-color experiments – Overcome the Complexity
http://www.fluorish.com/ http://www.biolegend.com/panelselector
http://www.ebioscience.com/resources/fluorplan-spectra-
viewer.htm
http://www.beckmancoulter.com/wsrportal/wsr/re
search-and-discovery/products-and-services/
flow-cytometry/research-tools/
index.htm
http://www.bdbiosciences.com/ecat/paneldesign
er.jsp
http://www.chromocyte.com/calculate
http://www.invitrogen.com/site/us/en/home/supp
ort/Research-Tools/Fluorescence-
SpectraViewer.html

Cell Cycle
 Figure from Purdue University Cytometry laboratories.
G1
M
G2
M (Mitosis)
Dividing the replicated chromosomes.
S G0
G1 (Gap1) Quiescent cells
interval between mitosis and
initiation of DNA replication,
RNA polymerases have access to the genome,
much protein synthesis.
G2 (Gap2)
interval between DNA
replication and mitosis
S
interval of time in
which the DNA is replicated

Cell Cycle
From Becton Dickinson

Cell Cycle
 DAPI – Excitation maximum = 358 nm,
Emission maximum = 461 nm
 DAPI is bound to dsDNA in AT clusters in
the minor groove.
 Because DAPI is excited by the violet
laser and emits in the blue wavelengths, it
is an excellent counter-stain for yellow,
green and red fluorochromes.

Cell Cycle
 Propidium Iodide – Excitation maximum =
493 nm, Emission maximum = 632 nm
 PI is bound to DNA by intercalating between
bases with no preference for purine or
pyrimidine base pairs. PI will also bind to
RNA.
 One PI molecule per 4-5 base pairs.
 PI cannot pass through intact cell
membranes, cells need to be dying or
permeabilized to allow PI staining.

Cell Cycle
Doublet Discrimination is very important with this technique!!!

Apoptosis
 Programmed Cell Death
 Characterized by DNA fragmentation and distinct changes in cell
morphology and volume.
 Requires biochemical energy.
 Important – For the normal functioning of the immune system,
embryonic development, normal tissue maintenance and
chemical- and hormone-induced cell death.
 ‘Programmed’-the genetically determined eradication of cells.
 Part of normal cell development, aging, and as a security
mechanism.
 Necessary and Pathological.

Apoptosis vs. Necrosis
 Necrosis
 Toxicity-induced cell death.
 Requires no energy, passive.
 Cells swell and then karyolysis (dissolution of the chromatin and
nucleus - DNase).
 Release of cellular contents may cause inflammation.
 Apoptosis
 ‘Stimulation’-induced cell death.
 Energy required.
 Cell shrinkage, then pyknosis (chromatin condenses), followed by
karyorrhexis (fragmentation of the nucleus).
 Do not release cellular contents and are readily phagocytosed by
macrophages.

Apoptosis
 Apoptotic Effects - Cell Morphology
 Cells change shape and shrink during apoptosis.
 The chromatin condenses in a process called Pyknosis.
 The cells become smaller and the cytoplasm shrinks around the
organelles.
Figure from the Cell
Migration Lab, University
of Reading
http://www.reading.ac.uk/c
ellmigration/apoptosis.htm

TUNEL Assay
 TUNEL (Terminal dUTP Nick-End Labeling)
 During Apoptosis, Genomic DNA is cleaved into small double-stranded
fragments and single-stranded breaks called ‘nicks’.
 Terminal deoxynucleotidyl transferase (TdT) labels DNA strand
breaks by catalyzing the polymerization of labeled nucleotides to
free 3’-OH DNA ends.
 The 3′-OH ends of the breaks can be detected by attaching a
fluorochrome. This is generally done directly or indirectly (biotin)
using fluorochrome-labeled deoxynucleotides in a reaction
catalyzed preferably by TdT.
 Best results are achieved using a positive control (fixed,
permeabilized cells treated with Dnase) and a negative control (no
FITC labeling reagent).
 We have had good luck with the Roche kit (cat # 11 684 795 910).

TUNEL Assay
Gated on Sperm Negative Control
Tunel FITC, No PI Positive Control
No Tunel, PI Only Positive Control Tunel FITC and PI Positive Control Tunel FITC and PI Test

Annexin V Assay
 Timeline
 1990 Andree at al. found that a protein, Vascular Anticoagulant ,
bound to phospholipid bilayers in a calcium dependent manner.
Protein was renamed Annexin V.
 1992 Fadok et al. discovered that macrophages specifically
recognize phospatidylserine (PS) that is exposed on the surface of
lymphocytes during the development of apoptosis. This PS is
normally on the inner leaflet of the membrane.
 1994 Koopman et al. developed a flow cytometric assay for
measuring FITC conjugated Annexin V binding to apoptotic cells.
Stained control and serum starved cells with ethidium bromide and
Annexin V-FITC.

Annexin V Assay
= Phosphatidylserine
Normal Cell Membrane
No PS on surface.
Apoptotic Cell Membrane
PS on surface.
Apoptotic/Necrotic Cell
Membrane PS on surface,
membrane disintegrates.

Annexin V + PI Apoptosis Assay
Annexin V binds to Phosphatidylserine on the Cell Membrane, PI to DNA

Flow Cytometry Assays
Immunophenotyping
DNA cell cycle/tumor ploidy
Cell tracking and proliferation
Cell Viability, Apoptosis, Necrosis
Fluorescent Protein expression
Cell Sorting
Cell Counting and Antigen quantification
Membrane and mitochondrial membrane potential
Intracellular protein staining
pH changes - BCECF
Redox state - NADH
Chromatin structure – Acridine Orange or 7-AAD
Total protein – Low MW dyes that bind to charged groups on proteins
Lipids – Nile Red
Surface charge – Fluoresceinated polycations
Membrane fusion/runover – MC540
Enzyme activity – Caspase, lysosomal, kinases etc
Sulfhydryl groups/glutathione – Oxidative metabolism, Fluorescien-5-
maleimide
DNA synthesis – Mitotic index
DNA degradation – apoptosis-associated DNA degradation
Gene expression
RNA Content – Pyronin Y
Cell Activation

Kumc introduction to flow cytometry

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