Flow Cytometry

Fluorescence-Activated Cell Sorter (FACS)

The fluorescence-activated cell sorter (FACS) is a powerful instrument used in flow cytometry to physically separate and identify types of cells from heterogeneous populations. (1,5,6,7,8) Flow cytometry can be used in various types of surface analysis and immunophenotyping, including platelet associated immunogloublining, studying platelets. Once labeled, the cells are forced to flow rapidly and single-file through the FACS instrument where they are excited by light from a focused laser beam. Each cell scatters some light and the labeled cells generate fluorescence signals from the dye. These two parameters are gauged by photodetectors, and the FACS computer uses them to make a decision to apply an electrostatic charge to cells with positive fluorescent intensities. The now charged single drops containing the cells of interest emerge from a nozzle and pass between two high voltage deflection plates and are thereby separated into a collection tube from the other unmarked cells. Up to 30,000 cells per second can be discriminated by the computer in modern sorters (1800000 cells per minute - over two directions, left and right)(changed through magnetism incorporation - cell sorting).

In ad DNA histogram cells are stained with a DNA-binding dye, such as propidium iodide. The DNA histogram can yield estimates for the number of cells in G1/G0, S and G2/M phases of the cell cycle. Cell turnover is the time for a cell to leave (for example, dying) and be further replaced in a given period. Cell half-life is half the time required for (plural = half cells to disapper) a cell to disappear (from total). Cell lifespan is the time between cell creation and cell death. Human T cells probably live for 3-12 months (average) (different for memory, much longer and activated T cells, anergy, etc…). The lifespan of a T cell in a child is the same as in an octagenarian. However, an octagenarian would have more memory cells and therefore, have more longer living cells than a child (greater, longer time of exposure to antigens).

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

2. Delves, P. and Roitt, I. 1999. Encyclopedia of Immunology. Academic Press Inc., 2nd ed., San Diego, USA

3. 1994. Current Protocols in Molecular Biology. Volume 2. John Wiley & Sons Inc., USA

4. Cruse, J. and Lewis R. 1995. Illustrated Dictionary of Immunology. CRC Press Inc., USA

5. http://biology.berkeley.edu/crl/flowcytometry.shtml

6. Lodish, H., Baltimore, D., Berk, A., Zipursky, Matsudaira, and Darnell, J. 2000. Molecular Cell Biology. American Scientific Books, USA

7. www.spectracell.com/jana.html

8. Andrade, W., Johnston, M. and Hay, J.B. 1998. The relationship of blood lymphocytes to the recirclating lymphocyte pool. Blood 91: 1653-1661.