Flow Cytometry

AGUSTINA TRI ENDHARTI SSi.PhD

SSi.PhD..
Medical Faculty
Brawijaya University
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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

Cell

particles (cells) in suspension

characterization (physical and chemical):

Measure size and granularity
Detect the expression of molecules (e.g. receptors,
cytokines) using fluorochrome-conjugated
monoclonal antibodies
Differentiate between live and dead cells

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Example Channel Layout for

PMT
Laser-based Flow
Cytometry
4

Flow cell

PMT

Dichroic
Filters

PMT
2

Bandpass
Filters
PMT
1

Laser

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 Detector
Fluorescence

is a property of
a dye (fluorochrome) that is
conjugated to a monoclonal
antibody that will bind to the
antigen/molecule of interest

Cell
Cell expressing
molecule of interest

The

fluorescence emitted by
each fluorochrome is detected
in a unique fluorescence
channel
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APC :Allophycocyanin
FITC :Fluorescein isothiocyanate
PE :Phycoerythrin
PerCP: Peridinin-chlorophyll-protein complex
Single-parameter histograms

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Analyze
Incubate

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Wash

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Analyze
Incubate

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Wash

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Applying Gates for sub-population analysis

CD3

Simple gating stratagies

Whole blood light scatter

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Gate on lymphocytes
(light scatter)

CD4
Assess T-cell population
(fluorescence)

Size and granularity using flow cytometry

Side
scatter

Forward
scatter

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Immunology/Haematology

Identification & classification of cells (cell surface

and/or intracellular antigens)

Cytopathology

DNA ploidy, Cell Cycle Analysis

Transfusion/Transplantation

Serology
HLA antibodies, cross-matching

Microbiology

Bacteria, virus-infected cells

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Detection Intracellular cytokine

Data.001

Data.001

40%

IFNg

IL-2

25%

100

101

102
103
FL2-Height CD4

104

100

101

102
103
FL3-Height CD4

104

CD4
Intracellular cytokine staining was performed to examine
production of IFN- CD8CD122 T cells express IFN-

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(Endharti, 2011. Journal of Immunology)

Cell Cycle Analysis

The cell cycle
DAPI
4',6 Diamidino-2-phenylindole dihydrochloride
Hoechst

UV

Propidium iodide (PI)

7-AAD
}

488

BrDU
Bromo-2'-deoxy-uridine

} 633

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Determine Its Position In The Cell

Cycle Based On Its DNA Content
G0-G1

G2-M

Events

Fluorescence Intensity
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In The Cell Cycle Based On Its DNA Content

Stromal
90.9%
0.62%
1.43%

IL-7
85.67%
2.64%
1.26%

The percentage of cells in G0/G1, S and G2/M phases of the cell

cycle
(Endharti, 2005. European Journal of Immunology)
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Assessing cell proliferation using flow cytometry

CFSE loaded cells

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Cell Proliferation
122-+sup122-

122-+sup122+

Counts

85.38%

45.26%
M1

M1

CFSE

The percentage of proliferated cells with reduced CFSE

(Carboxyfluorescein Diacetate Succinimidyl Ester )
fluorescence is shown in each panel.
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(Endharti, 2005. Journal of Immunology)

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Flow surface staining of fresh blood

1.
2.
3.
4.

Isolate PBMCs from 3ml of human blood

Collect whole blood in EDTA-anticoagulant tube
Label each tube (depend on group number)
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.

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6. Using a sterile pipet, remove the upper layer that contains the
plasma and most of the platelets. Using another pipet, transfer the
mononuclear cell layer to another centrifuge tube.
7. Wash cells by adding 10ml PBS and centrifuging 10 min at 1300
rpm, rt. Repeat washing process 2 times. Remove supernatant
completely, remain pellet only.
8. Add the following flow antibodies to the tube

PerCPPE
PBSCy5.5
2%FCS
CD3
CD14
100 l
Tube 1 (5L)
(5L)
Add 30 l staining solution above
Stain the cells for 20 minutes at room temperature in the dark
Add 400L flow buffer and vortex
Agustina`14 10,000-20,000 events on the flow cytometer
Acquire

3. Incubate for 20 minutes at room temperature in the dark

Antibodies will
bind to their
antigens

M
Gran

B
T
T

CD8-PE
CD4-FITC

Gran

This set of antibodies should bind to CD4+ or CD8+ T-cells

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