Simultaneous flow cytometric measurement of viability and lymphocyte subset proliferation

Combined analysis of DNA content and immunofluorescence on single cells by flow cytometry provides information on the proliferative response of cellul ar sub-populations in mixed cell preparations. However, the presence of con siderable numbers of dead (nonviable) cells impairs accurate flow cytometri c data analysis, mainly, because dead cells can bind antibodies non-specifi cally and show alterations in their DNA staining profiles. We developed a r apid method for identification of dead cells by fluorescence in cell prepar ations that are: stained simultaneously for two-color immunofluorescence an d DNA content. Cells are stained with 7-aminoactinomycin D (7-AAD) for dead cell discrimination and with fluorescein-isothiocyanate (FITC) and phycoer ythrin (PE)-labeled monoclonal antibodies (mAb) for cell surface immunofluo rescence. Diffusion of 7-AAD from stained, dead cells into unstained, live cells after cell permeabilization is blocked by the addition of its non-flu orescent analogue actinomycin D (AD). DNA is stained with red-excitable TO- PRO-3 iodide (TP3) which has an emission spectrum that can be effectively s eparated from the emissions of FITC, PE, and 7-AAD. TP3 staining is perform ed in the presence of ribonuclease A (RNAse) in phosphate-citrate buffer co ntaining saponin (PCBS) at low pH. FITC fluorescence is sensitive to acid p H: therefore, PCBS is replaced after DNA staining with 1X PBS at pH 7.2 con taining saponin to permit accurate detection of FITC immunofluorescence on the flow cytometer. We apply this method to the analysis of differential pr oliferation of lymphocyte subsets in cultures of human peripheral blood mon onuclear cells (PBMC) with low viability. (C) 2001 Elsevier Science B.V. Al l rights reserved.