What Is Flow Cytometry?
for Microbiology 542, Immunology Laboratory
Copyright © 2003 by Eric Martz
University of Massachusetts, Amherst MA US

Flow cytometers (FC or FCM) are automated instruments that quantitate properties of single cells, one cell at a time. They can measure cell size, cell granularity, the amounts of cell components such as total DNA, newly synthesized DNA, gene expression as the amount messenger RNA for a particular gene, amounts of specific surface receptors, amounts of intracellular proteins, or transient signalling events in living cells. Quantities are usually relative, but can be numbers of molecules per cell when absolute values are needed. Typically, up to three to six properties or components are quantitated in a single sample, cell by cell, for about 10,000 cells, in less than one minute (not counting time to prepare the sample, which might be an hour or more).

Simply measuring cell size and granularity is sufficient to distinguish the major categories of leukocytes in peripheral blood. This is the basis for clinical instruments that do automated complete blood counts (CBC). Adding fluorescent probes to the cells enables quantitation of specific structures ("flow cytofluorometry"). The most common use of flow cytofluorometry is for total DNA per cell in biopsy specimens from tumors, for clinical cancer diagnosis and prognosis. Another major use is quantiation of CD4+ vs. CD8+ T lymphocytes in blood to determine when an HIV infection has resulted in AIDS, and the degree to which anti-HIV drugs are working.

Flow cytometry is widely used in research. More than one third of papers in the Journal of Immunology include flow cytometric data, as do a substantial percentage of papers on cell structure, function, and mechanism in other journals.

Flow cytometers take in a suspension of monodisperse (single, unclumped) cells and run them one at a time (single file) past a laser beam. As each cell passes through the laser beam, scattered and fluorescent light are quantitated. Sensitivity is limited by "autofluorescence", naturally fluorescent components of cells that set a background fluorescence intensity -- fluorescent probes must emit substantially greater intensities in order for their signals to be quantitated accurately. Most flow cytometry is analytical: after the information is obtained as it passes through the cytometer, the sample is discarded. Some flow cytometry is preparative: living cells are sorted into separate containers based on the properties of each cell.

Flow cytofluorometry (FC) can be contrasted with fluorescence microscopy (FM). FC can quantitate total amounts of a component per cell for a large number of cells (typically 10,000, up to 100,000 easily). FC cannot ordinarily locate where a component is within the cell. FM shows whether a fluorescent component is uniformly distributed in the cell, or concentrated in anatomical compartments, and whether the distribution changes with time. FM can quantitate total amounts, and amounts in different anatomical compartments within cells, but typically for tens to hundreds of cells rather than the tens of thousands easily processed by FC. FC requires a monodisperse suspension of single (unclumped) cells, while FM does not. FM can provide information about cell interactions, while FC can rarely do this. FC can sort thousands of living cells according to their fluorescent properties, while FM cannot.

The objectives of the flow cytometry experiment in this class include:

  1. Identification of lymphocyte subpopulations
    1. obtaining percentage of cells with each type of receptor
    2. obtaining relative numbers of receptors per cell
  2. Relating the results to structure and function of the immune system

    General Scientific

  3. Working with living mammalian lymphocytes
  4. Understanding fluorescence and its power in scientific investigation
  5. Experience with computer data analysis software and graphics
  6. Experience with analysis of large amounts of data (four parameters times 10,000 cells = 40,000 numbers per sample)
  7. Understanding how flow cytometry works, its strengths and weaknesses
  8. Being able to interpret flow cytometric data in scientific publications