Flow Cytometry
FACS
(Fluorescence-Activated Cell Sorter)

Flow Cytometry
Leonard Herzenberg
ntechnique for analyzing populations of cells
ncells are measured individually but in large numbers
ncells are incubated with fluorescently labeled monoclonal antibodies directed against different
antigens (cell surface, intracellular, nuclear)
nCD numbers (clusters of differentiation)
nCD1-CD364 (Nov 2014)
nLineage specific markers
nUsed in phenotyping (leukaemias)
n

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CD-2
nezralé T-lymfocyty
CD-3
všechny T-lymfocyty (součást TcR), kromě NK buněk
CD-4
helperské T-ly
CD-7
T-ly nacházející se v thymu
CD-8
cytotoxické T-ly
CD-14
monocyty a makrofágy
CD-15
neutrofily, eosinofilní granulocyty
CD-16
NK buňky, neutrofily
CD-19
B-lymfocyty
CD-34
lymfoidní a myeloidní progenitorové buňky
CD-38
plazmatické buňky
CD-40
B-ly (izotypový přesmyk za přítomnosti CD-40 ligandu)
CD-45
panleukocytární antigen
CD-56
NK buňky
CD-58
endothelie, buňky prezentující antigen T-lymfocytům (adhese s CD-2)
CD-64
Fc γ receptor (makrofágy, neutrofily…)
CD-68
dendritické buňky
CD-80 a 86
buňky prezentující antigen T-lymfocytům
CD-95
FAS receptor
CD-203
bazofilní granulocyty

Blood October 15, 1997 vol. 90 no. 8 2863-2892
Common phenotypes and AML


Flow Cytometer Instrumentation
nfour general components
nFluidics
nOptics
nDetectors
nElectronics
nUnderstanding how a flow cytometer operates is critical to the design of your experiments

Flow Cytometry Fluidics
nThe cell sample is injected into a stream of sheath fluid
nLabeled cells are accelerated and individually pass through a laser beam
nits resulting fluorescence and angle deflection detected by a photocell
n

Flow Cytometer Optics
nLight emitted from the interaction between the cell and the laser beam is collected by a lens
nThe light moves through a system of optical mirrors and filters
nSpecified wavelengths are then routed to optical detectors

What can you measure with a Flow
Cytometer?
n1. Size and complexity of cells
nForward-scattered light (FSC)
nis proportional to the surface area or size of a cell
nSide-scattered light (SSC)
nis proportional to the granularity or internal complexity of a cell
n2. Cell surface molecules
n3. Nuclear antigens
n4. Protein expression/localization
n5. Copy number variation (Flow-FISH)
n6. Cell pigments
n




Fluorochrome Emission
nThe laser beam excites the fluorochrome at a specific wavelength and the fluorochrome emits light
at a separate wavelength (emission)
nProperty of any fluorescent dye; 2 things:
nExcitation spectra
nEmission spectra
n
nIf your laser functions at 488nm, find dyes that have excitation spectra at that λ
n

Flourochromes Have Overlapping Emission
Wavelengths


nDetection Systems
nPhotomultiplier Tubes (PMTs)
nHistorically 1-2
nCurrent Instruments 3-9
nIllumination Systems
nLasers (Blue 488 nm, Green 532 nm, Red 640 nm, violet 405 nm)
nBD FACS Calibur
nArgon ion (488)
nHeNe (633)
nBD FACS Canto II
nSolid state (488)
nHeNe (633)
nViolet (405)
nFACS Aria sorter
n
n
Technical Components

Electrostatic Flow Sorting



How do we detect the signal?
nPhotomultiplier tubes (PMT) and lenses
nFL-1 lets light through at 500-560 nm
nFL-2 lets light in at 560-611 nm
nFL-3 610-660 nm
nFL-4 >660 nm
nPicking dyes too close in λ results in no detection

Flow Cytometer Electronics
nThe voltage pulse height, width, and area are determined by the particle’s size, speed, and
fluorescence intensity
nparameters are acquired and analyzed in real-time by a computer

Antibodies (immunoglobulins)
nClone
nIsotype
nMouse, rat, rabbit, goat
nReactivity
nSpecificity
nFluorochrome
nCatalogue number and cost: $$$ £££ €€

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46_1



49_1



Polyclonal vs Monoclonal antibodies



Buňka 2755



Buňka 2762



17-01_WholeBloodSep_1
-Cells incubated at 4°C, 37°C or room temperature
-avoid non-specific binding
-always have a negative control

Flow Cytometry (Data)



Flow Plots (1D histogram, 2D scatter plots, 2D contour plots)



Gating



Application of FC
nPhenotyping
nCell function; proliferation, apoptosis, cell cycle
nIntracellular staining; IFNg
nNuclear staining
nClinical
ndetection of malignant cells
n