This work was performed in the UCLA Jonsson Comprehensive Cancer Center Flow Cytometry Laboratory and was supported by National Institutes of Health awards. CA-16042, CA-42735, AI-72656 and AI-72631 and by the American Cancer Society award CH-439.
Article first published online: 8 MAR 2005
Copyright © 1991 Wiley-Liss, Inc.
Volume 12, Issue 3, pages 279–285, 1991
How to Cite
Schmid, I., Uittenbogaart, C. H. and Giorgi, J. V. (1991), A gentle fixation and permeabilization method for combined cell surface and intracellular staining with improved precision in DNA quantification. Cytometry, 12: 279–285. doi: 10.1002/cyto.990120312
This work was presented in part at the XIVth Analytical Cytology Conference in Asheville, North Carolina, in march 1990.
- Issue published online: 21 JUN 2005
- Article first published online: 8 MAR 2005
- Manuscript Accepted: 10 DEC 1990
- Manuscript Received: 14 AUG 1990
- Flow cytometry;
- propidium iodide;
- internal antigens
A method was developed for gentle fixation of mammalian cells and permeabilization of their membranes. The method is useful for staining of intracellular antigens or quantification of DNA content simultaneously with cell surface staining. Cells are treated for 1 h at 4°C with 0.25% buffered paraformaldehyde then for 15 min at 37°C with 0.2% Tween 20 detergent in PBS. The procedure permits excellent staining of intracellular proteins, very low coefficients of variation (CV) on the G0G1-peak of DNA distributions, and preservation of the integrity of cell surface antigens. The low vs. 90° angle light scatter profile of cell clusters is maintained thereby allowing discrimination of different cell populations including human peripheral blood lymphocytes and monocytes for gating and analytic purposes. The method was successfully used on a variety of other cell types, including human thymocytes, murine thymocytes and spleen cells, and several leukemic cell lines. Dual-color surface antigen staining combined with DNA staining with 7-amino-actinomycin D (7-AAD) on peripheral blood mononuclear cells (PBMC) cultured with tetanus toxoid allowed the determination of the cell subset that was preferentially stimulated. Staining for internal antigens was done on CCRF-CEM for expression of CD3ϵ and on NALM-6 for expression of μ. The technique we developed gave bright and specific staining of internal antigens in the examples presented here. It is particularly suited for correlations of internal antigen staining with DNA staining and/or surface immunofluorescence.