A streptavidin-biotin-based three-step immunolabeling protocol for quantitative staining of intracellular antigens for flow cytometric analysis was evaluated using simian virus 40 (SV40) large T antigen. The concentration as well as the quantity of antibody used required optimization. The optimum labeling conditions varied moderately with cell lines that express T antigen levels over a 40-50-fold range. The procedure resulted in specific fluorescence 2.4 times higher than that using a comparable two-step indirect immunofluorescence technique. The gain in resolution was shown to be greater when staining cells with lower antigen levels. In the analysis of back-ground fluorescence, the principal components were, as for the two-step technique, autofluorescence and propidium spectral overlap. While streptavidin does add to the background, the increase is relatively small. Decreasing the propidium concentration from 50 p g / d to 5 pg/ ml was found to reduce significantly the level of background from this source. Theoretical aspects of quantitative staining and of resolution versus quantification are discussed. o 1992 Wiey-Liss, Inc.
Keyterms: SV40, large T antigen, indirect immunofluorescence, propidium iodide, multiparameter analysis 'This work was supported by NIH grants IIL419S and CA43703.