This work was supported by NIH grants HL419S and CA43703
Streptavidin-based quantitative staining of intracellular antigens for flow cytometric analysis†
Article first published online: 8 MAR 2005
Copyright © 1992 Wiley-Liss, Inc.
Volume 13, Issue 7, pages 711–721, 1992
How to Cite
Srivastava, P., Sladek, T. L., Goodman, M. N. and Jacobberger, J. W. (1992), Streptavidin-based quantitative staining of intracellular antigens for flow cytometric analysis. Cytometry, 13: 711–721. doi: 10.1002/cyto.990130707
- Issue published online: 21 JUN 2005
- Article first published online: 8 MAR 2005
- Manuscript Accepted: 15 MAR 1992
- Manuscript Received: 27 NOV 1991
- large T antigen;
- indirect immunofluorescence;
- propidium iodide;
- multiparameter analysis
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 background 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 μg/ml to 5 μg/ ml was found to reduce significantly the level of background from this source. Theoretical aspects of quantitative staiaing and of resolution versus quantification are discussed. © 1992 Wiley-Liss, Inc.