Flow cytometry

Flow cytometry is a technique for counting, examining and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of single cells flowing through an optical/electronic detection apparatus.

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A beam of light (usually laser light) of a single frequency (colour) is directed onto a hydrodynamically focused stream of fluid. A number of detectors are aimed at the point where the stream passes through the light beam; one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter (SSC) and one or more fluorescent detectors). Each suspended particle passing through the beam scatters the light in some way, and fluorescent chemicals in the particle may be excited into emitting light at a lower frequency than the light source. This combination of scattered and fluorescent light is picked up by the detectors, and by analysing fluctuations in brightness at each detector (one for each fluorescent emission peak) it is possible to deduce various facts about the physical and chemical structure of each individual particle.

Related Topics:
Laser - Hydrodynamically focused - Scattered - Fluorescent

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FSC correlates with the cell volume and SSC depends on the inner complexity of the particle (i.e. shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness).

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Measurable parameters are:

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• volume and morphological complexity of cells
• cell pigments
• DNA (cell cycle analysis, cell kinetics, proliferation etc.)
• RNA
• chromosome analysis and sorting (library construction, chromosome paint)
• proteins
• cell surface antigens (CD markers)
• intracellular antigens (various cytokines, secondary mediators etc.)
• nuclear antigens
• enzymatic activity
• pH, intracellular ionized calcium, magnesium, membrane potential
• membrane fluidity
• apoptosis (quantification, measurement of DNA degradation, mitochondrial membrane potential, permeability changes)
• cell viability
• monitoring electropermeabilization of cells
• oxidative burst
• characterising multi-drug resistance (MDR) in cancer cells
• glutathione
• various combinations (DNA/surface antigens etc.)

This list is very long and constantlty expanding.

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Flow cytometry: Flow cytometers ►