unit 6.2

There are four basic methods for staining cells with antibodies for immunophenotyping by flow cytometry. The first method (see Basic Protocol 1) is an indirect one that employs a primary unconjugated monoclonal antibody followed by a secondary, fluorochrome-conjugated polyclonal antibody. Because the target antibody is not conjugated with a fluorochrome in this method, a second fluorochrome-conjugated polyclonal antibody— derived from a different animal species and directed against the IgG from the species that generated the first antibody—is used. An intact polyclonal antibody preparation should never be used for immunophenotyping; the fluorochrome-conjugated F(ab')2 fragment should always be used (see Critical Parameters).

The second method (see Alternate Protocol 1) is also an indirect method, in this case employing a hapten-conjugated primary antibody followed by a fluorochrome-conju-gated polyclonal antibody against the hapten. Examples of haptens are digoxigenin, dior trinitrophenol, and sometimes biotin. A variation on this method included in Alternate Protocol 1 employs a biotinylated antibody followed by a fluorochrome-conjugated streptavidin. The fourth method (see Alternate Protocol 2), which is the method of choice, employs a directly conjugated monoclonal antibody against the desired antigen.

Combinations of the above methods are also described for two-color staining (see Basic Protocol 2 and Alternate Protocols 3 and 4) as well as three- and four-color staining (see Alternate Protocols 5 to 9). The ethidium monoazide (EMA) procedure for detecting nonviable cells in a cell population is also included (see Support Protocol 1). Finally, analysis of the data acquired from flow cytometry using cells stained by the above procedures is detailed (see Support Protocols 2, 3, and 4).

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