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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