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 particles (cells) in suspension

Cellcharacterization (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 4

Cytometry
Dichroic PMT
Filters 3
Flow cell
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 Cell
antigen/molecule of interest
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 Wash

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 Analyze

Incubate Wash

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Applying Gates for sub-population analysis
Simple gating stratagies

CD3
CD4
Gate on lymphocytes Assess T-cell population
Whole blood light scatter
(light scatter) (fluorescence)

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 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

25% 40%

IL-2
IFNg

100 101 102 103 104 100 101 102 103 104
FL2-Height CD4 FL3-Height CD4

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
S
Events

Fluorescence Intensity
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 In The Cell Cycle Based On Its DNA Content

Stromal IL-7
90.9% 85.67%

0.62% 2.64%

1.43% 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+

85.38% 45.26%
Counts

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. Isolate PBMCs from 3ml of human blood
2. Collect whole blood in EDTA-anticoagulant tube
3. 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.

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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

PerCP- PE PBS-
Cy5.5 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
Acquire
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3. Incubate for 20 minutes at room temperature in the dark

Antibodies will
bind to their T
M
antigens
T Gran

T
B
T

CD8-PE T M
CD4-FITC Gran

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

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