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What is flow cytometry?
Flow cytometry is the analysis of individual particles, by detecting their light-absorbing or fluorescing properties as they pass single-file through a laser beam.
Particles must be single and individual; clumps of particles cannot be analyzed.
Light-scattering characteristics measure intrinsic properties of the particle, such as cell size (FALS) and cellular complexity (SS).
By adding fluorescent dyes a vast number of other parameters can be measured. Fluorescent dyes fall into at least three categories:
- Functional properties (e.g. Nile Red, a lipid specific probe; or Indo-1 for Ca++)
- Fluorescently-labeled antibodies
- Kinetic probes where a cellular constituent interacts with a non-fluorescent ligand to release a fluorochrome (e.g. CFDA releases fluorescein in live cells).
Multiple individual parameters can be measured on a single cell, then plotted into frequency histograms. Because all measurements are collected on each individual cell, this technique eliminates problems inherent in bulk techniques. Bulk techniques, such as spectrophotometry and colorometric analysis, rely on population averages to describe cells when in truth this "average" may be derived from bi- or tri-modal distributions.
Examples of variables that can be measured:
- Cell size and granularity
- DNA and RNA content
- Cytoplasmic and surface receptors
- Intracellular ion levels (Ca++, Zn++, Cl-, Mg++)
- Lectins
- Membrane potential (mitochondrial and cytoplasmic)
- Nuclear antigens
- Enzyme kinetics can also be measured, using time as a parameter.