Nuclei were isolated from various cell types including Chinese hamster ovary (CHO) and L1210 leukemia cell lines, primary cultures of fibroblasts, nonstimulated and stimulated human lymphocytes and mouse liver cells, by using different isolation techniques. The isolated nuclei were subsequently stained with acridine orange (AO) and their fluorescence was measured by flow cytometry. Various procedures designed to stain DNA uersus RNA differentially with A 0 were tested, and the staining of isolated nuclei was compared with that of whole cells. Control incubations with RNase and DNase were performed to estimate in whole cells and in nuclei the contribution of DNA and RNA to the fluorescence intensity at the respective wavelength bands of maximum emission for DNA (F530) and RNA (F,6tn).
Depending on the cell type, 10-20% of total cell RNase-sensitive F,600 is localized in the nuclei. The RNase-resistant portion of F,600 of isolated nuclei represents the stainability of DNA. Suppression of cell proliferation in subconfluent cultures results in a decrease in both whole cell and in nuclear RNA content. Nonstimulated lymphocyte nuclei have considerably lower RNA content than nuclei from lymphocytes stimulated by pokeweed mitogen. Two subpopulations of nuclei having the same (2C) DNA content but differing in RNA content, are present in mouse liver; the cells entering S phase originate from the high RNA population.
Key terms: Acridine orange, cell cycle, metabolic compartments, cycling and quiescent cells
Flow cytometric techniques were developed to measure DNA and RNA content simultaneously in whole cells (6)(7)(8)24). These techniques, when applied in a variety of cell systems, provide useful information regarding cell metabolism during the cell cycle, differentiation, or quiescence (reviews, 8, 11,13). Because cellular RNA content varies in different types of neoplastic cells and often is different in normal uersus tumor cells, the DNA/RNA measurements have also found an application in clinical cytology (1,18,20).
In the present study, the technique of DNA/RNA measurements was applied to isolated cell nuclei. Several different cell types were examined and the data on isolated nuclei were