Cell cycle-related changes in nuclear chromatin of stimulated lymphocytes as measured by flow cytometry

Flow cytometric techniques have been developed to assay lymphocyte stimulation as reflected by the increase in the cell transcriptional activity and cell progression through the cell cycle. The metachromatic fluorescent dye, acridine orange, is used to (a) stain DNA and RNA differentially in individual cells, and (b) stain nuclear chromatin after removal of cellular RNA BY RNase and cell pretreatment at acidic pH. Stimulated cells with diploid DNA content (G1) have an increased content of stainable RNA that makes it possible to distinguish them from nonstimulated (G0) cells. G0 cells can also be distinguished from G1 cells based on differences in stainability of their nuclear chromatin after treatment with acid. Mitotic indices can be scored automatically, inasmuch as the metaphase chromatin stains differently than does chromatin in the interphase cells. Altogether, the numbers of cells in the G0, G1, S, G2, and M phases may be obtained rapidly and with great accuracy. The cell transciptional activity can be correlated with changes in nuclear chromatin (e.g., during the transition from G0 to G1). The two independent techniques may also prove to be useful in recognizing and quantitating noncycling cells in other cell systems. The possible mechanisms responsible for differential stainability of nuclear chromatin in cells at different phases of the cell cycle are discussed.