Flow cytometry Principles and Applications

1. Flow cytometry- Principles and
Applications
Sanju Kaladharan
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2. • Flow cytometry is a technique used to detect and measure physical
and chemical characteristics of a population of cells or particles
• A sample containing cells or particles is suspended in a fluid and
injected into the flow cytometer instrument.
• The sample is focused to ideally flow one cell at a time through a
laser beam and the light scattered is characteristic to the cells and
their components.
• Cells are often labeled with fluorescent markers so that light is first
absorbed and then emitted in a band of wavelengths.
• A flow cytometry analyzer is an instrument that provides
quantifiable data from a sample. Other instruments using flow
cytometry include cell sorters which physically separate and
thereby purify cells of interest based on their optical properties.
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4. • Flow cytometry is a popular cell biology technique that utilizes
laser-based technology to count, sort, and profile cells in a
heterogeneous fluid mixture.
• Using a flow cytometer machine, cells or other particles suspended
in a liquid stream are passed through a laser light beam in single file
fashion, and interaction with the light is measured by an electronic
detection apparatus as light scatter and fluorescence intensity.
• If a fluorescent label, or fluorochrome, is specifically and
stoichiometrically bound to a cellular component, the fluorescence
intensity will ideally represent the amount of that particular cell
component.
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5. • Three main systems make up the flow cytometer instrument
and those are
 the fluidics,
 the optics and
 the electronics.
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6. The fluidic system
• The purpose of the fluidics system is to transport the particles in a
stream of fluid to the laser beam where they are interrogated.
•
• Any cell or particle that is 0.2 to 150 μms in size can be analyzed. If the
cells are from solid tissue, they require disaggregation before they can be
analyzed.
• Although cells from animals, plants, bacteria, yeast or algae are usually
measured, other particles such as chromosomes or nuclei can also be
examined.
• Some particles such as marine algae are naturally fluorescent, but in
general, fluorescent labels are required to tag components of the particle.
• The section of the fluid stream that contains the particles is referred to as
the sample core.
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7. • When a cell suspension is run through the cytometer, sheath fluid is
used to hydrodynamically focus the cell suspension through a small
nozzle.
• The tiny stream of fluid takes the cells past the laser light one cell at
a time .
• Light scattered from the cells or particles is detected as they go
through the laser beam.
• A detector in front of the light beam measures forward scatter (FS)
and several detectors to the side measure side scatter (SS).
• Fluorescence detectors measure the fluorescence emitted from
positively stained cells or particles.
• Sheath fluid focuses the cell suspension, causing cells to pass
through a laser beam one cell at a time. Forward and side
scattered light is detected, as well as fluorescence emitted from
stained cells.
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9. Antibody staining Methods
Direct staining
•In direct
immunofluorescence
staining, cells are incubated
with an antibody directly
conjugated to a fluorophore
such as Peridinin
Chlorophyll Protein Complex
(PerCP).
• It is advantageous during
intracellular staining
because large antibody-
fluorophore complexes
including secondary
antibodies can become
trapped and result in non-
specific binding, or they
may fail to enter the cell
which results in no
detection.
Indirect Staining
• In indirect staining, the
fluorophore conjugated
secondary antibody
detects the primary
antibody which is
unconjugated.
• Another available
method is the avidin-
biotin system, whereby a
biotin-conjugated
antibody is detected with
fluorophore-labeled
avidin.
Intracellular
staining:
•allows direct measurement
of antigens (cytokines or
transcription factors)
present inside the cell
cytoplasm or nucleus
without tissue seperation.
•Detecting intracellular
antigens requires cell
permeabilization before
staining, and antibodies
should be prepared in
permeabilization buffer to
ensure the cells remain
permeable.
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10. The optics system
• The optics system is made up of lasers which illuminate the particles
present in the stream as they pass through and scatter light from the
laser.
• Any fluorescent molecules that are on the particle emit fluorescence,
which is detected by carefully positioned lenses.
• Generally, the light scattered from up to six or more fluorescences
is determined for two different angles.
• Optical filters and beam splitters then direct the light signals to the
relevant detectors, which emit electronic signals proportional to the
signals that hit them.
• Data can then be collected on each particle or event and the
characteristics of those events or particles are determined based on
their fluorescent and light scattering properties.
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12. • Cells or particles passing
through the beam scatter
light, which is detected as FS
and SS.
• FS correlates with cell size and
SS is proportional to the
granularity of the cells.
• In this manner, cell
populations can often be
distinguished based on
differences in their size and
granularity alone.
The direction of light scattered by the
cell correlates to cell size and granularity
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13.  Eg :
 FITC (fluorescein
isothiocyanate) channel
PMT will detect light
emitted from FITC at a
wavelength of
approximately 519 nm.
 The PE channel PMT will
detect light emitted from PE
(phycoerythrin) at 575 nm
wavelength.
 Each PMT will also detect
any other fluorochromes
emitting at a similar
wavelength to the
fluorochrome it is detecting.
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14. When no fluorescing
cells pass through the
optics,
no photons are
emitted and no signal
is detected.
As the fluorescent
labeled cell passes
through
the optics and is
interrogated by the
laser, photons are
emitted and
so the intensity of the
voltage measured
increases.
As each fluorescing
cell completes its path
through the laser
beam, this leaves a
pulse of voltage over
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15. The electronics system
• The electronics system is used to change the light signals detected
into electronic pulses that a computer can process.
• The data can then be studied to ascertain information about a large
number of cells over a short period.
• Information on the heterogeneity and different subsets within cell
populations can be identified and measured.
• Some instruments have a sorting feature in the electronics system
that can be used to charge and deflect particles so that certain cell
populations can be sorted for further analysis
• The data are usually presented in the form of single parameter
histograms or as plots of correlated parameters, which are referred
to as cytograms.
• Cytograms may display data in the from of a dot plot, a contour
plot or a density plot. sis 15SANJU KALADHARAN

16. • A useful example of this is when
running blood samples on the flow
cytometer.
• Larger and more granular granulocyte
cells produce a large population with
high SS and FS.
• Monocytes are large cells, but not so
granular, so these produce a separate
population with high FS but lower SS.
• Smaller lymphocytes and
lymphoblasts produce a separate
population with less FS. They are not
granular cells, so also have low SS.
• Therefore, these cells can be
separated into different populations
based on their FS and SS alone.
Each dot represents a single cell
analyzed by the flow cytometer
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17. Applications
• Immunophenotyping- Using fluorescence-conjugated antibodies
directed toward a protein(s) of interest, cells expressing that
protein(s) on the surface or intracellularly may be detected by flow
cytometry.Specific cell types may be distinguished within a mixed
population using multiple fluorescence-conjugated antibodies.
• Transfection efficiency may be determined when a fluorescent
protein (i.e. GFP) is used as a marker.
• Apoptosis measurement- Several flow cytometric methods to
detect apoptosis are available. Cells can be stained with Annexin V
or 7ADD.
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18. • Cell cycle analysis- Fixed cells are stained with a dye that binds to
DNA (e.g. propidium iodide,ethidium bromide,DAPI).
• Fluorescent intensity is used to determine the amount of cellular
DNA present in each cell (i.e. two copies of a genome have roughly
twice the fluorescent intensity of one copy).
• Cell proliferation- Carboxyfluorescein diacetate, succinimidyl ester
(CFSE) is a dye that diffuses into cells and is passed from parent to
daughter cells so that the cells of each generation have half the
fluorescent intensity of their parent cells.
• Cell sorting- A particular subset(s) of cells may be sorted from a
mixture of cells based upon particular properties.
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19. • Membrane potential- Bacterial membrane potential may be
analyzed using DiOC2, which exhibits green fluorescence in all
bacterial cells, but shifts to red fluorescence as the dye becomes
more concentrated in cells with larger membrane
potentials.Mitochondrial membrane potential may be analyzed in
the same manner with JC-1.
• Live/dead bacteria discrimination- You can test how fast an
antibiotic is killing microbes: live cells have intact membranes and
are impermeable to dyes such as propidium iodide, which only
leaks into cells with compromised membranes. Thiazole orange
enters all cells, live and dead, to varying degrees. Thus a
combination of these two dyes provides a rapid and reliable
method for discriminating live and dead bacteria.
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21. Thank You
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