Flow cytometric analysis of double-stranded RNA content distributions

Abstract

A staining procedure is described for quantitative analysis of cellular RNA content distributions by flow cytometry (FCM). Cells were fixed in ethanol, treated with DNAse and stained with propidium iodide. FCM analysis showed that more than 90% of the fluorescence resulted from the double-stranded RNA (dsRNA) bound fluorochrome. The dsRNA content distributions were measured in HeLa S3 cells in culture 24-120 hr after plating, in L1210 ascitic leukemic cells 1-7 days after transplantation, and in regenerating bone marrow 3-7 days after injection of cyclophosphamide. In all three cell populations, maximal dsRNA content was observed during the period of most active cell proliferation, as determined by the S-phase index derived from the DNA histograms. In the dsRNA distributions in L1210 leukemia cell populations 1-2 days after transplantation, two separate peaks were evident, representing weakly fluorescent, presumably nontumor cells, and intensively fluorescent tumor cells. The amount of dsRNA-bound fluorochrome was significantly greater in L1210 cells than in normal and regenerating bone marrow cells and also greater in spontaneous thymic lymphoma cells than in normal thymic cells.