Flow CytometryFlow Cytometry(Principles and Main Applications)

Hossein Asgarian-OmranHossein Asgarian-OmranPh.D., Immunology

Dep. of Immunology, School of MedicineMazandaran University of Medical SciencesMazandaran University of Medical Sciences

asgarianhossein@yahoo.com

Workshop objectives

Getting familiar with flow cytometry system

p j

Getting familiar with flow cytometry system

How to interpret flow cytometry-related results andHow to interpret flow cytometry related results andgraphs in scientific documents

How to use this system in your own projects

HistoryThe first fluorescence‐based flow cytometry device (ICP 11):by Wolfgang Göhde (University of Münster, Germany. 1968)

The first commercialized by German developer andmanufacturer Partec in Göttingen (1968‐69)g ( )

The first FACS instrument from Becton Dickinson (1974)

Original name of flow cytometry technology was: "pulsecytophotometry"cytophotometry

8 years later in 1976, at the Conference in Florida, the namewas changed to "flow cytometry", a term that quicklybecame popular.

Definition

Flow Cytometry

Flow Cytometry is the process whereby such measurements aremade upon cells/particles as they pass through a measuring apparatussuspended in a fluid stream.

FACS

Fluorescence Activated Cell SorterFluorescence Activated Cell Sorter

Basics of Flow CytometryBasics of Flow CytometryBasics of Flow CytometryBasics of Flow Cytometry

Cells in suspension

flow in single file throughFluidicsFluidics

flow in single-file through

an illuminated volume where they

scatter light and emit fluorescence

that is collected filtered and

OpticsOpticsthat is collected, filtered and

converted to digital valuesElectronicsElectronics

that are stored on a computerElectronicsElectronics

SampleFluidicsFluidicsSheath

Cells are presented t th l i

Flow chamber

to the laser using principles of hydrodynamichydrodynamic focusing

Laser optics

L BLaser Beam

Optical DesignOptical Design

PMT 6

PMT 5

DichroicFiltersFl ll

PMT 4Sample

PMT 2

PMT 3FiltersFlow cell

Scatter PMT 2

BandpassFilters

Laser Scatter

SensorPMT 1

Optical Filters

Dichroic Filter/Mirror at 45 degg

Transmitted LightLight Source Transmitted LightLight Source

Reflected light

Types of Optical Filters

LONG (700nm)

Wavelengths

Short Pass Long Pass Band Pass

550 Long Pass (650LP)

650 Short Pass (600SP)

Pass Through Filters

600/100 Band Pass (600/100)

Short Pass Long Pass Band Pass

(650LP)(600SP)SHORT (500nm) (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

550 Long Pass (650LP)

650 Short Pass (600SP)SHORT (500nm)

Filters

Transmitted <650 nm >550 nmDiflected 90° >650 nm <550 nm

FL3

Optical Bench Schematic

FL1 S

FL2 Sensor 575BP

Sensor 620BP

FL4 Sensor 675BP

SS Sensor

Sensor 525BP

Fluorescence Pickup Lens

600DL

645DL

Laser Beam

488DL

488BK

550DL

Flow Cell

FS Sensor

488DL

From Fluorescence to Computer Display

• Individual cell fluorescence quanta is picked up by the variousdetectors (Photo Multipliers Tubes, PMT’s).detectors (Photo Multipliers Tubes, PMT s).

• PMT’s convert light into electrical pulses and amplify them.

• These electrical signals are digitized using Analog to DigitalConverters (ADC’s).

• Different parameters are determined for each single event.

Different  Parameters Measured by Flow Cytometryy y y

Intrinsic Extrinsic

• No reagents or probes  • Reagents are required.

Intrinsic Extrinsic

g prequired (Structural)

ll i ( d i h

‐ Mostly fluorescent probes

– Cell size (Forward Light Scatter)

– Cytoplasmic grabularity (90 d Sid Li h S )degree Side Light Scatter)

Forward Angle Light Scatter or FSCForward Angle Light Scatter or FSC

Laser

FALS Sensor

Laser

90 Degree Light Scatter or SSC

FALS Sensor

Laser

90LS Sensor

Fluorophores in Flow Cytometry

Common FluorophoresCommon Fluorophores

• Organic: FITC, APC, PE, PerCP

• Tandem: PECY7, PECY5.5, APC‐tandem 

• Nanocrystal: Q‐dot(Invitrogen), eFluor y g(eBiosciences) and Brilliant Violet (Biolegend and BD Biosciences)

Common Fluorophores in Flow Cytometry u p w y y

400 nm 600 nm 700 nm

ty

Wavelength

500 nm

E it ti

e In

tens

it ExcitationEmission

Rel

ativ

e

Fluorescein (FITC)Fluorescein (FITC)

Fluorochromes (cont…)

W l th

Fluorochromes (cont…)

400 nm 600 nm 700 nm

ity

Wavelength

Excitation500 nm

ve In

tens

i

Emission

Rel

ativ

Phycoerythrin (PE)Phycoerythrin (PE)

• Each cell generates a quanta of fluorescencePhotomultiplier Tubes

(PMT’s)

PE FL FITC FL 488nm Sct

Discriminating Filters

Confocal LensDichroic Lenses

FiltersForward Light Scattering Detector

Common Laser LinesCommon Laser Lines

PEPE--TR ConjTR Conj600 nm300 nm 500 nm 700 nm400 nm

457350 514 610 632488

Texas RedTexas Red

PEPE--TR Conj.TR Conj.

PIPI

EthidiumEthidium

PEPEPEPE

FITCFITC

ciscis--Parinaric acidParinaric acid

THE WALTER AND ELIZA HALL INSTITUTE OF MEDICAL RESEARCH

Common Fluorochromes:FITC and Phycoerythrin

FITC

400 450 500 550 600 650 700

R_PhycoErythrin (PE)

400 450 500 550 600 650 700

THE WALTER AND ELIZA HALL INSTITUTE OF MEDICAL RESEARCHFluorochrome Combinations: 3 lasers, 11 colour immunofluorescence

Cascade Blue

Cascade Yellow 545/90

440/40

,

FITC

PE 575/25

525/50

PE.Cy5

PE.Cy5.5 695/40

665/30

PE.Cy7

TexasRed 625/40

784/45

APC Cy7

APC.Cy5.5

APC

750LP

705/50

660/40

Wavelength (nm)350 400 450 500 550 600 650 700 750 800 850

APC.Cy7 750LP

Conjugation of antibodies with flourophoresConjugation of antibodies with flourophores

Panel DesigningPanel Designing

• Instrument configuration andInstrument configuration and characterization

• Dye selection based on lasers and filtfilters

• Proper reagent selection

Different Display Graphsp y p

2 Parameters Dot PlotSingle Positive PI Population

Double Positive Population

PE FL

Negative Population

FITC FL Single Positive FITCFITC Population

2 Parameters Dot Plot

104

0.01 69.910 4

0.24 7.24

10 2

10 3

L2-a

bTC

R P

E

10 2

10 3

FL2-

Vd2

PE

0

10 1FL2-

H: F

L

7 5422 6

10 1

FL2-

H: F

10 0 10 1 10 2 10 3 10 4

FL1-H: FL1-CD3 FITC

10 0 7.5422.6

10 0 10 1 10 2 10 3 10 4

FL1-H: FL1-Vg9 FITC

10 0 1.3991.1

1 Parameters Histogram

M1M1

Fluorescence intensity

FITC

vent

s

FITC

FITC

FITC

mbe

r of E

v FITC

FITC

Num

101 104103102

Relative fluorescence intensityRelative fluorescence intensity

1 Parameter Histogram

Positive

N tiNegative

BrighterDimmerCount

4

6

1

1 2 3 4 6 7 150 160 170 .. 190

Channel NumberFluorescence picked up from the FITC PMT

Why Look at FSC v. SSCy• Since FSC ~ size and SSC ~ internal structure, a correlated

t b t th ll f diff ti ti f llmeasurement between them can allow for differentiation of cell types in a heterogeneous cell population

LymphocytesGranulocytes

SSC

Lymphocytes

Monocytes

RBCs, Debris,RBCs, Debris,Dead CellsDead Cells

FSC

Gating in Flow Cytometryg y y• A gate is a numerical or graphical boundary that can

be used to define the characteristics of particles tobe used to define the characteristics of particles toinclude for further analysis

LymphocytesGranulocytes

SSC

Lymphocytes

Monocytes

FSC

Different Gating Strategies in Flow CytometryDifferent Gating Strategies in Flow Cytometry

PolygonPolygonRegion or rangeQuadrant

M1 10 3

104

R P

E

0.01 69.9

10 1

10 2FL

2-H

: FL2

-abT

C

10 0 10 1 10 2 10 3 10 4

FL1-H: FL1-CD3 FITC

10 0 7.5422.6

Compensationp

Th t i t tThe most important challenge in g

multi-color flow cytometry

Fluorescence Activated Cell SortingFluorescence Activated Cell Sorting

Cell SortingCell Sorting

488 nm laser FALS Sensor

Fluorescence Activated Cell SortingFluorescence Activated Cell Sorting

488 nm laser FALS Sensor

Fluorescence detector

+-Ch d Pl t +Charged Plates

Single cells sortedinto test tubes

Flow Cytometry in Different EraFlow Cytometry  in Different Era

• Immunology and hematologygy gy

• Cell Kinetics

• Genetics

• Molecular Biology

• Pharmacology 

• Microbiology

• Parasitology

• Animal Husbandry (and Human as well)• Animal Husbandry (and Human as well)

• Biological Oceanography

• BioterrorismBioterrorism

Applications of Flow CytometryDetermination of cellular PhenotypesAbsolute cell countingImmunophenotyping of leukemia and lymphomaImmunophenotyping of leukemia and lymphomaMeasurement of intracellular and nuclear antigens (cytokines)Functional assays by flow cytometry (phagocytosis) Analysis of cell division using CFSEAnalysis of cell division using CFSEAnalysis of reticulocyte Staining of microbial cellsDNA analysisChromosome analysis and sortingApoptosisApoptosisFlow cytometric screening of cell‐based librariesMultiplexed particle based flow cytometric assaysCell sortingCell sortingand …..

Functional Assays by Flow CytometryFunctional Assays by Flow Cytometry

• Phagocytosis• Oxidative metabolism• Hydrogen peroxide production• Glutathione levels• Degranulation assays• Ion flux measurement of cytosolic free Ca• Detection of dead cells and measurement of cell killikilling

ImmunophenotypingImmunophenotyping 

• Detecting the origin/stage of differentiation ofDetecting the origin/stage of differentiation of leukemia/lymphoma

• Detecting early recurrence of hematological g y gmalignancies

• Diagnosis/monitoring inherited/congenital g g gimmunodeficient patients

• Chemotherapeutic monitoring

• Diagnosis/monitoring autoimmune disease

• Pre/post transplantation monitoring/evaluationp p g

DNA AnalysisDNA Analysis

GG22MM GG00

DNA AnalysisDNA AnalysisGG11

ssGG00GG11ss

Cou

ss GG22MMnt

0 200 400 600 800 1000

DNA content22NN 44NN

DNA content

Staining of microbial cells•Real time analysis •Measurement of total cell count•Monitoring of microorganisms in food, water and …•Sorting and identifications of bacterial spores

B.anthracis

B.subtilisSS

irradiated B.anthracis

SS

FS

Reticulocyte Analysis

•Erythropoietin therapeutic effect•BMT recovery

50

BMT recovery•Evaluation of erythropoiesis

15

112

Cou

nt

75

RR11RR22RR33RR44

1000

0

37

Thiazole Orange.1 1000100101

Apoptosis

Dual Staining with Annexin V and PIua Sta g t e a d

Multiplexed particle based flow t t icytometric assays

-The technology utilizes microspheres as the solid support for a conventional immunoassay affinity assay or DNA hybridization assayconventional immunoassay, affinity assay or DNA hybridization assay.

Ab against microorganismsCytokine

Various protein DNA and RNAVarious protein, DNA and RNAViral antigens

VWF…….

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