Flow cytometry is a laser-based technique to count and analysis the size, shape and properties of individual cells within a heterogeneous population of cells.
2. Introduction
Flow cytometry is a laser-based technique to count and analysis the size, shape
and properties of individual cells within a heterogeneous population of cells.
Flow cytometry is a widely used approach to phenotype the cells and to
assessing the purity of isolated subpopulations.
Flow cytometry data is extremely quantitative, it provides fast, objective and
simultaneous multi-parameter analysis of single cells as well as physical
separation of cells of particular interest.
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3. Introduction
It is measured by the fluorescence intensity resulted from
fluorescent-conjugated antibodies directly recognizing target
proteins, or ligands specifically binding to molecules within a cell.
A heterogeneous mixture of biological cells can be sorted into two
or more containers by using a specialized type of flow cytometry
called fluorescence-activated cell sorting (FACS).
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4. Flow Cytometer
Suspensions of individual cells from cultured cells, tissues
and organisms are prepared in tubes and then rapidly placed
on a flow cytometer.
Flow cytometer is constituted of five main components.
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6. When each cell passes the laser beam,
the laser beam is scattering in all
directions.
By analyzing the data of forward and side
scatter together, cell populations can
often be distinguished based on
differences in their size, shape and
internal complexity.
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Measurement of Forward and Side Scatter
7. Flow cytometry provides cell phenotyping by using fluorescent-
labeled antibodies directly against surface markers.
Flow cytometry executes intracellular analysis with antibodies or
ligands detecting multiple intracellular targets including proteins and
nucleic acids.
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Measurement of Fluorescence Signaling
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Staining
Direct staining is recommended for intracellular staining, where
large complexes like secondary antibodies are difficult to enter
the cell, which causing non-specific binding or failure of primary
antibody detection.
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Fluorochromes
Highly expressed antigens will usually be detected and easy to
be resolved from the negative control with almost any
fluorochrome.
While, antigen with lower expression level require the higher
signal to background ratio provided by a brighter fluorochromes
to separate the positive cells adequately from the unstained
cells.