Flow Cytometry Medical Faculty Brawijaya University AGUSTINA TRI ENDHARTI AGUSTINA TRI ENDHARTI SSi.PhD SSi.PhD. Agustina`14
Jan 05, 2016
Flow Cytometry
Medical Faculty Brawijaya University
AGUSTINA TRI ENDHARTI AGUSTINA TRI ENDHARTI SSi.PhDSSi.PhD..
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Flow refers to a fluid stream
Cyto refers to a cell
metry refers to measurement.
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Simultaneous measurements of multiple characteristics of a single cell
Measurements are made on a per-cell basis at routine rates of 500 to 4000 cells per second
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Enumerate particles (cells) in suspension
Cell characterization (physical and chemical):Measure size and granularityDetect the expression of molecules (e.g. receptors,
cytokines) using fluorochrome-conjugated monoclonal antibodies
Differentiate between ‘live’ and ‘dead’ cells
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PMT
PMT
PMT
PMT
DichroicFilters
BandpassFilters
Example Channel Layout for Laser-based FlowCytometry
Laser
1
2
3
4
Flow cell
original from Purdue University Cytometry Laboratories; modified by R.F. Murphy
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Light source is focused on single file of cells
Excitation optics (lasers):A laser is used to provide a single wavelength of light. Multiple lasers can be installed to provide coincident light of different wavelengths
Collection optics (detectors):Emitted light from fluorochromes is directed to appropriate detectors. Light from forward scatter, side scatter and emitted fluorescence are also detected
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This is how a whole blood sample appears under flow cytometry analysis
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Fluorescence is a property of a dye (fluorochrome) that is conjugated to a monoclonal antibody that will bind to the antigen/molecule of interest
The fluorescence emitted by each fluorochrome is detected in a unique fluorescence channel
Cell
Cell expressing molecule of interest
Fluorescence Detector
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APC :AllophycocyaninFITC :Fluorescein isothiocyanatePE :PhycoerythrinPerCP: Peridinin-chlorophyll-protein complex
Single-parameter histograms
Analyze
WashIncubate
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Analyze
WashIncubate
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Applying Gates for sub-population analysisSimple gating stratagies…
Whole blood light scatter Gate on lymphocytes(light scatter)
Assess T-cell population(fluorescence)
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CD
3
CD4
Forward scatter
Side scatter
Size and granularity using flow cytometry
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Immunology/HaematologyIdentification & classification of cells (cell surface and/or intracellular antigens)
CytopathologyDNA ploidy, Cell Cycle Analysis
Transfusion/Transplantation SerologyHLA antibodies, cross-matching
MicrobiologyBacteria, virus-infected cells
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CD4
IFN
gDetection Intracellular cytokine
Intracellular cytokine staining was performed to examine production of IFN- CD8CD122 T cells express IFN-
(Endharti, 2011. Journal of Immunology)
100 101 102 103 104FL2-Height CD4
Data.001
100 101 102 103 104FL3-Height CD4
Data.001
IL-2
25% 40%
Cell Cycle Analysis
DAPI4',6 Diamidino-2-phenylindole dihydrochloride
Hoechst } UV
Propidium iodide (PI)7-AAD } 488
BrDUBromo-2'-deoxy-uridine } 633
The cell cycle
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G0-G1
SG2-M
Fluorescence Intensity
Even
ts
Determine Its Position In The Cell Cycle Based On Its DNA Content
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Stromal90.9%
0.62%
1.43%
IL-785.67%
2.64%
1.26%
The percentage of cells in G0/G1, S and G2/M phases of the cell cycle
In The Cell Cycle Based On Its DNA Content
(Endharti, 2005. European Journal of Immunology)
Assessing cell proliferation using flow cytometryCFSE loaded cells
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M1
122-+sup122- 122-+sup122+
Cell Proliferation
CFSE
Cou
nts
M1
85.38% 45.26%
(Endharti, 2005. Journal of Immunology)
The percentage of proliferated cells with reduced CFSE (Carboxyfluorescein Diacetate Succinimidyl Ester ) fluorescence is shown in each panel.
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1. Isolate PBMCs from 3ml of human blood2. Collect whole blood in EDTA-anticoagulant tube3. Label each tube (depend on group number)4. Prepared 15 ml conical centrifuge tubes. Using a sterile pipet,
add an equal volume (3ml) Ficoll-Hypaque at room-temperature. Whole blood put on layer of the Ficoll-Hypaque slowly.
5. Centrifuge 30 min at 1000 rpm , rt.
Flow surface staining of fresh blood
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6. Using a sterile pipet, remove the upper layer that contains theplasma and most of the platelets. Using another pipet, transfer themononuclear cell layer to another centrifuge tube.
7. Wash cells by adding 10ml PBS and centrifuging 10 min at 1300rpm, rt. Repeat washing process 2 times. Remove supernatantcompletely, remain pellet only.
8. Add the following flow antibodies to the tube
PerCP-Cy5.5
PE PBS-2%FCS
Tube 1CD3(5µL)
CD14(5µL)
100 µl
•Add 30 µl staining solution above•Stain the cells for 20 minutes at room temperature in the dark•Add 400µL flow buffer and vortex•Acquire 10,000-20,000 events on the flow cytometer
3. Incubate for 3. Incubate for 20 20 minutes at room temperature in the dark minutes at room temperature in the dark
T
Antibodies will bind to their antigens
CD8-PE
CD4-FITC
M
M
T
TTT
T
B
Gran
Gran
This set of antibodies should bind to CD4+ or CD8+ T-cellsAgustina`14
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