Flow cytometry is a laser-based technique that quantitatively analyzes individual cells in a heterogeneous population, allowing for rapid, simultaneous multi-parameter assessments. It involves sorting cells using fluorescence-activated cell sorting (FACS) and requires a flow cytometer composed of key components such as a light source and optical system. The technique employs fluorescent-labeled antibodies for cell phenotyping and intracellular analysis, with two types of staining methods: direct and indirect.

1. Flow Cytometry Guide
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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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5.  Light source;
 Flow chamber;
 Optical system;
 Detectors;
 Computer.
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Flow Cytometer

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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Measurement of Fluorescence Signaling
Fluorescence intensity measurement.

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Measurement of Fluorescence Signaling
Example of multiple fluorochromes detecting.

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Staining
 Single-cell suspension is
prepared in staining buffer and
then cells are incubated with
fluorochromes.
 There are two types of staining:
direct and indirect staining.

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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.

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14. Thanks
For more info about flow cytometry and antibodies,
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