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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
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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
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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.
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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
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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
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SampleFluidicsFluidicsSheath
Cells are presented t th l i
Flow chamber
to the laser using principles of hydrodynamichydrodynamic focusing
Laser optics
L BLaser Beam
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Optical DesignOptical Design
PMT 6
PMT 5
DichroicFiltersFl ll
PMT 4Sample
PMT 2
PMT 3FiltersFlow cell
Scatter PMT 2
BandpassFilters
Laser Scatter
SensorPMT 1
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Optical Filters
Dichroic Filter/Mirror at 45 degg
Transmitted LightLight Source Transmitted LightLight Source
Reflected light
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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
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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
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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.
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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)
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Forward Angle Light Scatter or FSCForward Angle Light Scatter or FSC
Laser
FALS Sensor
Laser
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90 Degree Light Scatter or SSC
FALS Sensor
Laser
90LS Sensor
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Fluorophores in Flow Cytometry
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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)
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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)
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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)
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• 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
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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
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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
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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
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Conjugation of antibodies with flourophoresConjugation of antibodies with flourophores
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Panel DesigningPanel Designing
• Instrument configuration andInstrument configuration and characterization
• Dye selection based on lasers and filtfilters
• Proper reagent selection
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Different Display Graphsp y p
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2 Parameters Dot PlotSingle Positive PI Population
Double Positive Population
PE FL
Negative Population
FITC FL Single Positive FITCFITC Population
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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
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1 Parameters Histogram
M1M1
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Fluorescence intensity
FITC
vent
s
FITC
FITC
FITC
mbe
r of E
v FITC
FITC
Num
101 104103102
Relative fluorescence intensityRelative fluorescence intensity
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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
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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
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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
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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
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Compensationp
Th t i t tThe most important challenge in g
multi-color flow cytometry
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Fluorescence Activated Cell SortingFluorescence Activated Cell Sorting
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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
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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
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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 …..
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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
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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
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DNA AnalysisDNA Analysis
GG22MM GG00
DNA AnalysisDNA AnalysisGG11
ssGG00GG11ss
Cou
ss GG22MMnt
0 200 400 600 800 1000
DNA content22NN 44NN
DNA content
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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
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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
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Apoptosis
Dual Staining with Annexin V and PIua Sta g t e a d
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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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