Kumc introduction to flow cytometry

Introduction to Flow

CytometryRich Hastings

KUMC Flow Cytometry Core Lab

3901 Rainbow Boulevard

Kansas City, KS 66160

913-588-0627

[email protected]

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 Cytometry1930’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.

The History of Flow Cytometry

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

Injector

Tip

Sheath

fluid

(PBS)

The principal of hydrodynamic focusing confines cells to the core (sample) stream by a cell-free sheath fluid.

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.

How Flow Cytometers WorkSheath pressure is constant.

Sample pressure is variable.

Flow is laminar.

Flow cell shape allows for

hydrodynamic focusing.

Hydrodynamic focusing

causes cells to line up

along their long axes.

Aria LSR II


From BD LSR II manual

Higher Flow Rate = Good for Qualitative Measurements

Lower Flow Rate = Greater Resolution and Quantitative Measurements


Laser is an acronym forLight 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.


From: http://www.antonine-education.co.uk

Electromagnetic Spectrum

Violet Laser Blue Laser Red Laser


Laser

interrogates

Cell in Flow Cell

Laser interrogation of a cell tells us physical properties of that 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

NeutrophilMonocyte


Pulse Processing

From BD LSRII Manual

Laser


Parameters Measured

From BD LSRII Manual

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.


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).


Figures from Flow Cytometry - A Basic Introduction

by Michael G. Ormerod

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.


Figure from Jane Limer BD Application Scientist

Tandem Dyes – One molecule is excited by laser light and donates the energy

to the acceptor molecule.


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.




CellForward light scatter 0ºA measure of cell size.

Side scatter 90ºA measure of cell granularity.

488 nm



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 DataFlow 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

DoubletsDoublet

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 CycleV

olt

ag

e In

ten

sit

y

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

CellsA 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

CompensationUncompensated Data

Voltages

PE = 538

PerCP = 791

APC = 690

Compensated Data

Comp MatrixPerCP -%PE = 22.01

APC - %PE = 0.09

APC - %PerCP = 4.54

PerCP - %APC = 1.04

CompensationUncompensated Data

Voltages

PE = 650

PerCP = 825

APC = 725

Compensated Data

Comp MatrixPerCP -%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.

FlowJo Compensation

FlowJo Compensation

CompensationAnnexin-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 StatisticsFor 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

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

Cell Staining Overview

Immunophenotyping-Antibody

Dilution

Figure from Flow Cytometry - A Basic Introduction

by Michael G. Ormerod

Most antibody manufacturers advise a dilution to start with.

We advise performing a dilution curve.

Immunophenotyping-Dilution

Protocol

32.4%, Mean = 3886

2.14%, Mean = 1042

1.4%, Mean = 886

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

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+.

ImmunophenotypingMulti-color experiments – Overcome the Complexity

Setting up an Experiment

From Lora Barsky, USC Flow Core

ImmunophenotypingMulti-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

http://www.fluorish.com/

http://www.biolegend.com/panelselector

http://www.ebioscience.com/resources/fluorplan-spectra-viewer.htm

http://www.beckmancoulter.com/wsrportal/wsr/research-and-discovery/products-and-services/flow-cytometry/research-tools/index.htm

http://www.bdbiosciences.com/ecat/paneldesigner.jsp

http://www.chromocyte.com/calculate

http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.html

Cell Cycle

Figure from Purdue University Cytometry laboratories.

G1

MG2

S G0

M (Mitosis)Dividing the replicated chromosomes.

Quiescent cellsG1 (Gap1)interval between mitosis and

initiation of DNA replication,

RNA polymerases have access to the genome,

much protein synthesis.

S interval of time in

which the DNA is replicated

G2 (Gap2)interval between DNA

replication and mitosis

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

http://www.reading.ac.uk/cellmigration/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 AssayGated on Sperm Negative Control

Tunel FITC and PI Positive ControlNo Tunel, PI Only Positive Control Tunel FITC and PI Test

Tunel FITC, No PI Positive Control

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

Cell Sorting

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

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