Flow Cytometry

Flow Cytometry

A powerful tool for defining, characterizing and enumerating cells, lymphocytes, is the flow cytometer, which detects and counts individual cells passing in a stream through a laser beam. A flow cytometer quipped to separate the identified cells is call a fluorescence-activated cell sorter (FACS). These instruments can study the properties of cell subsets identified using monoclonal antibodies to cell-surface proteins. Individual cells within a mixed population are first tagged by treatment with specific antibodies labeled with fluorescent dyes, or by specific antibodies followed by labeled anti-Ig. The mixture of labeled cells is then forced with a much larger volume of saline through a nozzle, creating a fine stream of liquid containing cells spaced singly at intervals. As each cell passes through a laser beam, it scatters the light and any dye molecules bound to the cell will be excited and will fluoresce. Sensitive photomultiplier tubes detect both the scattered light, which gives information on the size and granularity of the cell, and the fluorescence emissions, which give information on the binding of the labeled monoclonal antibodies and therefore, on the expression of cell-surface proteins by each cell. In addition, flow cytometry can be modified to sort and sequence subsets of cell populations. These are called cell sorters, and they do this by passing a flowing stream over a piezoelectric crystal, which vibrates at high frequency.

References

1. Janeway, C.A., Travers, P., Walport, M., and Capra, J.D. 1999. Immunobiology: The immune system in health and disease. Garland Publishing, 4th ed., New York, USA