Analysis of intracellular antigens by flow cytometry

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Abstract
Quantitative immunofluorescence is routinely used in flow cytometric assay of cell surface antigens. Intracellular antigens have not been as tractable. Recent publications (Proc Natl Acad Sci 80:5573–5577, 1983; Cytometry 6:208–214, 1985) and the results presented here demonstrate that highly specific staining and subsequent quantitative analysis are not only possible but rather easily achieved. High purity antibodies and optimized fixing and staining technique are required. Under conditions presented in this paper, 97% of fluorescein specific signal is specific to the T antigen of SV40 when monoclonal antibody to this antigen is used with a transformed cell line. Three levels of quantitative analysis are discussed: 1) estimation of the fraction of positive cells in a mixed +/− population, 2) estimation of the average content of antigen in a population of cells, and 3) measurement of the distribution of antigen content within a population of cells. Results are presented that demonstrate that relatively low specific signal (measured as percentage of total signal) can be tolerated to achieve the first level and that the current methods available that produce a high specific signal are sufficient to achieve the second level. The third level will require further research aimed at lowering the variation introduced by the method of measurement.