Flourescence-Acativated Cell Sorting (FACS)

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TABLE OF
CONTENT

01 Introduction 05 Data analysis

Components of a flow
06 Common protocols
02 cytometer

Fluorochromes and their Applications of flow

03 07 cytometry
properties

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Fluorochromes used in 08 References
flow cytometry
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Principles of flow cytometry and FACS

Flow cytometry is a popular cell biology technique that utilizes laser-based

technology to count, sort, and profile cells in a heterogeneous fluid mixture.
If a fluorescent label, or fluorochrome, is specifically and stoichiometrically
bound to a cellular component, the fluorescence intensity will ideally represent
the amount3 of that particular cell component.

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 There are two different types of flow cytometry –non-sorting and
sorting. Non-sorting type can perform light scattering and
fluorescence emission while the sorting type has the ability to sort
particles as well. Fluorescent activated cell sorters (FACS) are flow
cytometers that have the capacity to sort fluorescent-labeled cells from
a mixed cell population.

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2. components of a flow cytometer

The main components of flow cytometers and cell sorters are basically
fluidics, optics (excitation and collection), an electronic network
(detectors) and a computer.

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 The fluidics is responsible for directing liquid containing
particles to the focused light source.
 The excitation optic focuses the light source on the
cells/particles while collection optics transmits the light scatter
or fluorescent light of the particle to an electronic network.

 The electronic network detects the signal and converts the

signals to a digital data that is proportional to light intensity.
 The computer is also required to analyze data.

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[Link] and their properties

The number of intrinsically fluorescent compounds in the cell is limited as well as its provided information.
Therefore, the cells are usually stained with fluorescent probes called fluorochromes that are able to show the
presence of components that otherwise would not be visible.
The important features of a fluorochrome include an absorption spectrum at which a fluorescent compound can
be excited and a range of emitted wavelengths called its emission spectrum. The emission wavelength of any
fluorochrome will always be longer than its excitation wavelength.

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[Link] used in
Fluorochromes used in flow cytometry are classified into several groups
flow cytometry including fluorochromes used to label proteins covalently,
fluorochromes for nucleic acids and reporter molecules.
the probe is commonly selected as an antibody. However, other proteins
such as a lectin, hormone, avidin or streptavidin, or even c-DNA may be
labeled using various fluorochromes. The most widely used
fluorochromes for labeling antibodies include FITC, phycoerythrin (PE)
and allophycocyanin (APC).

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FITC easily reacts with the amino groups on the lysine residues in the protein and produces
moderately stable conjugates.
FITC is a good fluorochrome for single-color staining since its maximum absorbance is near 490
nm. On the other hand, its emission with longer wavelengths makes it inappropriate for multicolor
applications.
its fluorescence is highly pH-sensitive and subjected to photobleaching with a high rate. To solve
these problems, various FITC derivatives have been developed with greater photostability and
increased fluorescence.

PE and APC are called phycobiliproteins that are the components of photosynthetic systems.
cells labeled with phycobiliprotein antibodies have fluorescence intensities between five- and 10-
fold greater than those labeled with FITC-labeled antibody.
Although using an argon laser excites FITC and PE, the excitation of APC needs helium–neon laser
due to its higher (650 nm) absorption maxima. The major drawback of using phycobiliproteins is
related to their higher molecular weight, causing steric changes when conjugated to proteins. They
can also give higher backgrounds if the cells are not washed properly.

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The development of tandem dyes, containing two Each fluorochrome has a wide emission spectrum resulting in
fluorochromes, has increased the number of labeled proteins some overlap between the fluorochromes when multiple
to be used. fluorochromes are used.
In tandem dyes, when the first dye is excited and reaches its This spectral overlap is corrected by subtracting a fraction of the
maximal absorbance, it transfers all its energy to the second FITC signal from the PE signal or vice versa. This process is
dye located in close proximity. As a result, this second named as color compensation.
fluorochrome is activated and produces the fluorescence
emission. This process is called fluorescence resonance
energy transfer (FRET).

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[Link] protocols Cell surface staining

Cell surface antigens localized on the plasma membrane of

The most common cell staining the cells are the most common proteins to use for flow
principles for identifying and cytometry to identify and characterize the cell types.
investigating functions of a single
cell are surface and intracellular
staining.

Intracellular antigen staining

Intracellular
12 proteins of cells can also be detected by flow

cytometry.

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[Link] analysis
The major principles of data analysis are to selectively show the cells of interest
and to find out more about your cells. This method is called ‘‘gating’’ in flow
cytometry.
a gate is a numerical or graphical boundary that can be used to define the
characteristics of particles for further analysis.
The most common application of gating strategy is to use FSC and SSC plots.

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 The data can be displayed by several different plot types. These range from histograms to
2-D plots such as dot plots, contour and density plots, to 3-D plots such as a tomogram
plot.

Histogram Density plot Dot plot

A histogram is a single parameter plot where These are graphs that display two measurement A dot plot provides a two-parameter display of
the x-axis represents the parameter’s signal parameters, one on the x-axis and one on the y- data and each dot represents one or more events
value in channel numbers and the y-axis axis and the cell count as a density (dot) plot or (particles) while density plot displays two
represents the number of events per channel contour map. parameters as a frequency distribution.
number. A dot plot provides a two-parameter display of The contour plot is the same as a density plot,
data and each dot represents one or more events except the density of the cells is displayed as
(particles) contour lines.
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[Link] of flow cytometry
 Phenotypic characterization of blood cells
 Measurement of apoptosis markers
 Cell viability
 Detection of plasma membrane changes
 Detection of active caspase-3 activity
 Detection of mitochondrial proteins
 DNA fragmentation
 Intracellular cytokine detection

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