Subcompartments of the G1 phase of cell cycle detected by flow cytometry.

Abstract

Cellular DNA and RNA were simultaneously quantitated in individual cells of cycling and quiescent populations by flow cytometry. Based on differences in RNA content, two distinct subcompartments, GIA and GIB, were recognized in the G1 phase of exponentially growing cells. After mitosis, cells reside in the low-RNA, GIA compartment from which they cannot enter the S phase directly. An increase in RNA above a critical level is required for G1 cells to be able to initiate DNA replication; G1 cells with RNA values above this level are classified as GIB. Cell transition from GIA to GIB and from GIB to S was analyzed in a stathmokinetic experiment on L1210 cells with a doubling time of 10 hr. The half-time of cell residence in the indeterminate state of G1 was found to be 1 hr, and the duration of the deterministic portion of G1 phase was 2 hr. described (8). Cells were studied either during the exponential phase of growth or during quiescence induced by serum deprivation, prolonged maintenance until confluence, or addition of sodium n-butyrate, as described in the figure legends. Other details related to the cell system and preparation of cells for measurements by flow cytometry are given elsewhere (2-6, 8).Cell Staining and Fluorescence Measurements. A detailed description of the technique is given elsewhere (2, 3). 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 MIM 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 maximal emission at 530 nm and interaction with RNA gives red metachromasia at 640 nm (2). The intensity of green fluorescence was linearly proportional to the content of DNA per cell within the range 2-93 pg (9) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Rapid analysis of drug effects on the cell cycle

“…As shown before 2. The G1 cell population may be further subdivided into two qualitatively distinct subcompartments, GIA and GIB differing in at as described (7,8). The threshold dividing the GI population into G l~ and G I R is a continuation of the right borderline of the S-cell cluster.…”

“…cycle compartments in a single measurement (5)(6)(7)(8). The results provided here are based on studies of a few selected drugs.…”

“…Fiuorescence measurements were performed within the first 10 min after cell staining, using an FC 200 Cytofluorometer (Ortho Diagnostic, Westwood, MA), as described (3)(4)(5)(6)(7)(8)13). Red (600-640 nm) and green (515-570 nm) fluorescence emissions from each cell, representing the extent of denatured and double-stranded DNA respectively (3), were measured simultaneously by separate photomultipliers and the data stored in the computer.…”

Flow cytometric discrimination of mitotic nuclei by right‐angle light scatter

“…Secondparameter measurements include forward light scatter, protein content (fluorescein isothiocyanate), RNA content (acridine orange, pyronin y), 5-bromodeoxyuridine (BudR) incorporation, and nuclear andor surf'ace antigens (2,4,6-8,10,12-16,18,23). The acridine orange (AO) fluorescence spectrum is different for single-stranded (red) and double-stranded (green) nucleic acids (7). Also, the nucleus exhibits structural differences in different stages of the cell cycle as reflected in its varying resistance to denaturation by acid or heat.…”

“…Also, the nucleus exhibits structural differences in different stages of the cell cycle as reflected in its varying resistance to denaturation by acid or heat. In conjunction with A 0 staining, these structural differences allow resolution of subclasses of the GI and G2 phases, as well as the M phase (6)(7)(8). In addition, the subclasses can be differentiated using two fluorochromes: propidium iodide (PI) to measure DNA content and fluorescein isothiocyanate (FITC) to measure protein content.…”