Flow cytometry is a technology that rapidly analyzes single cells or particles as they flow past single or multiple lasers while suspended in a buffered salt-based solution. Each particle is analyzed for visible light scatter and one or multiple fluorescence parameters.
It is a technique
which is used to detect, quantify and sort out the antigen present in a given
sample. The principle of flow cytometry is immunofluorescence in which
fluorescent molecules absorb light of one wavelength (excitation) and emit
light of another wavelength (emission). If antibody molecules are tagged with a
fluorescent dye, or fluorochrome, and excited by light of appropriate wavelength,
immune complexes containing these fluorescently labelled antibodies (FA) can be detected by coloured light emission.
Antibody molecules bound to antigens in cells or tissue sections can similarly
be visualized. Various techniques which use immuno-fluroscence are extremely
valuable qualitative tools, even though they do not give quantitative data.
This short coming was remedied by flow cytometry, which was designed to
automate the analysis and separation of cells stained with fluorescent antibody.
Flow cytometry uses laser beam
and light detector to count intact single cells in suspension. Every time, when
a cell passes the laser beam, light is deflected from the detector, and this
interruption of the laser signal is recorded. In those cells, having a fluorescently
tagged antibody bound to their surface, antigens are excited by the laser and
light is emitted. This emission is recorded by a second detector system,
located at right angle to the laser beam. The instrument counts each cell while
laser beam passes through it and records the level of fluorescence the cell
emits; an attached computer generates plots of the number of cells and their
fluorescence. More sophisticated versions of the instrument are capable of
sorting populations of cells into different containers according to their
fluorescence profile.
Applications
- Analysis of whole human blood for diagnosing diseases.
- Sorting different blood cell fractions for ex-vivo manipulations and/or transplantations.
- Immuno-phenotypic analysis of murine blood to identify transgenic/knockout animals.
- Sorting and analysis of a slew of cell lines for various biological assays.
- Characterization and isolation of rare cells types like adult stem cells and cancer initiating cells.
Cell sorting - Cell sorter is a specialized flow cytometer which can physically isolate the desired cells in separate collection tubes. The cytometer characterizes each cell as it passes through the laser. The sorter then identifies and "'kick the target cells into a test tube.
Cell cycle analysis - Flow çytometry can analyze cell replication using fluorescent dyes to measure the distinct phases of the cell cycle. It can also measure cell aneuploidy, associated with chromosomal abnormalities.
Apoptosis- Apoptosis, or programmed cell death, is a normal part of the life cycle of eukaryotic cells. Cells die for a variety of reasons, particularly by necrosis due to external physical and chemical changes, or by apoptosis in which cells initiate a "suicide" programme through internally controlled factors. These two distinct types of cell death, apoptosis and necrosis, can be distinguished by flow cytometry on the basis of differences in morphological, biochemical and molecular changes occurring in the dying cells.
Immunophenotyping- The commonest application of cytometry is immunophenotyping. This technique identifies and quantifies populations of cells in a heterogeneous sample - usually blood, bone marrow or lymph. In clinical labs, immunophenotyping is useful in diagnosing hematological malignancies such as lymphomas and leukemia.