Lymphocyte stimulation: a rapid multiparameter analysis.

Abstract

ABSIRACTWe propose a simple cytofluorometric assay of lymphocyte stimulation. It was developed from earlier efforts in this laboratory to differentially stain DNA and RNA in fixed cells by use of a metachromatic dye, acridine orange (4), and applied to distinguish stimulated from nonstimulated lymphocytes (5). Under appropriate conditions, acridine orange preferentially stains nucleic acids, among other cellular polyanions. The dye intercalates into the double helix as a monomer that fluoresces green (530 nm) (6), while electrostatic dye binding to the phosphates of single-stranded nucleic acids involves dye aggregation ("stacking") and dye-dye interactions that result in red fluorescence (640 nm) (7). To obtain differential stainability of DNA compared to RNA in situ, it is necessary to selectively denature any double-stranded RNA prior to staining (4, 5), because otherwise the binding of acridine orange to double-stranded RNA is indistinguishable from binding to native DNA (4). This is achieved by treatment of cells with the chelating agent EDTA, which is known to unfold ribosomes (8). MATERIALS AND METHODSHuman mononuclear cells were isolated from peripheral blood of normal volunteers by density centrifugation of Ficoll-Hypaque. The lymphocytes were separated from monocytes, and were cultured in the absence or presence of phytohemagglutinin, as described before (9 (10).Observation of cells under phase microscopy reveals that Triton X-100 does not lyse the cells; the cell membrane remains preserved, and the mitotic cells remain intact after the treatment with detergent at that low pH. Inspection of cells stained with.acridine orange under ultraviolet fluorescence microscopy indicates that the green fluorescence is localized exclusively in the cell nucleus, while the red fluorescence is confined to the cytoplasm and nucleoli. RESULTS AND DISCUSSIONThe present method involves initial treatment of unfixed cells with chelating agents (citrate, EDTA) in the presence of the nonionic detergent Triton X-100, at pH 3.0, followed by staining with acridine orange at pH 3.8. Control experiments on cells first treated with DNase or RNase (Table 1) reveal that the detergent treatment makes cells permeable not only to the dye but also to nucleases, and that at least 88% of the green fluorescence (Fmo) is the result of acridine orange interaction with cellular DNA, while at least 87% of the red fluorescence (F>600) of stimulated cells is due to RNA. This relatively good specificity in differential staining of DNA and RNA by acridine orange may be due to the following: (i) Exposure of permeable cells to chelating agents and the dye, as in the case of fixed cells (4, 5) or isolated ribosomes (8), denatures double-stranded RNA in situ. (i)0 Cell staining is done at pH 3.8, i.e., near optimal for the discrimination of DNA from RNA (11). In fact, morphological recognition of stimulated lymphocytes, based on RNA stainability with acridine orange, was shown to be optimal at pH 3.8 (12). (iii) The cells are stained in the presence of...

New cell cycle compartments identified by multiparameter flow cytometry

“…Normal human peripheral blood lyphocytes, quiescent and stimulated by a variety of mitogens (25,28,(30)(31)(32) and in mixed allsgeneic reactions (53).…”

“…Simultaneous Staining of RNA and DNA A detailed description of this technique is given elsewhere (15,20,22,24,25,52). Briefly, cells made permeable by Triton X-100 (Sigma) were stained with chromatographically purified acridine orange (Polysciences Inc., Warrington, PA) at a final dye concentration of 13 @ and dye/DNA-P molar ratio >2, in the presence of 1 mM Na-EDTA.…”

“…Under these conditions interaction of the dye with DNA results in green fluorescence with maximum emission at 530 nm while interaction with RNA gives red metachromasia at 640 nm, the intensity of which is proportional to RNA content (7). The specificity of staining was evaluated by treatment of cells with RNase A (Worthington Biochemical Corp., Freehold, NJ) or with DNase I (Worthington) as described (15,25,52).…”

“…In the first technique, after selective denaturation of double stranded RNA, cellular DNA and RNA are stained differentially allowing for discrimination between cells with different RNA content for any given DNA value (15,22,25,(51)(52)(53)(54). In the second technique, susceptibility of DNA in situ to denaturation is measured in cells pretreated with RNase and then ' Supported by US PHS Grants 1ROlCA23296 and 126CA14134.…”

“…Specifically, we have measured: (a) total content of DNA per cell (15,19,24,50,52); ( b ) content of DNA accessible to A 0 (22,24,25,52); (c) RNA content per cell (22,25,[51][52][53][54]; ( d ) sensitivity of DNA in situ to acid denaturation (19-23, 26-29, 44); (e) incorporation of BUdR (30); and ( f ) cell, or nuclear diameter (pulsewidth) (48). At least forty different cell types including normal and tumor cells in vivo and in vitro, as well as numerous cell lines at various phases of growth have been investigated.…”