Simultaneous staining of deoxyribonucleic (DNA) and ribonucleic acid (RNA) in nonfixed, but permeable, cells is described. Cells are made permeable by treatment with non-ionic detergent at low pH. RNA is denatured prior to, or during staining, by exposure of cells to chelating agents to ensure that DNA (native) and RNA (dentured) may be stained differentially with the metachromatic dye, acridine orange. The fluorescence of individual cells is measured in a flow cytofluorometer. A comparison between various staining procedures employing acridine orange or other intercalating dyes in unfixed cells is discussed in terms of staining specificity, cell permeability and preservation. Evidence is provided that acridine orange staining of unfixed cells may be used as a simple, fast means of obtaining information on cell ploidy levels and cell cycle status from DNA measurements (green fluorescence), and cell transcriptional activity from RNA staining (red fluorescence), in human and murine cells lines, peripheral blood and bone marrow specimens from patients with leukemia and mitogenically (phytohemagglutinin) or antigenically (mixed lymphocyte culture) stimulated human peripheral blood cultures. Exposure of cells to detergent at low pH as an alternative to cell fixation or hypotonic treatment is proposed as a fast, convenient method of making cells permeable to dyes.

Simultaneous staining of ribonucleic and deoxyribonucleic acids in unfixed cells using acridine orange in a flow cytofluorometric system.

A new method of testing for mitogen-induced lymphocyte stimulation: Measurement of the percentage of growing cells and of some aspects of their cell kinetics with an electronic particle counter

A flow-cytofluorometric method, based on the differential stability of deoxyribonucleic acid versus ribonucleic acid with the metachromatic dye, acridine orange, simultaneously measures the following parameters of stimulation in mixed lymphocyte cultures: (a) number of nonstimulated cells; (b) total number of stimulated lymphocytes; (c) number of stimulated lymphocytes in G1, S and G2 + M phases of the cell cycle; (d) number of macrophages; (e) number of dead cells. The progress of lymphocyte stimulation may also be measured by a parameter representing ribonucleic acid accumulation per cell. The method is rapid, avoids cell rinsing, fixation and centrifugation and is applicable to microcultures. Multiparameter analysis of cell stimulation which provides simultaneous measurements of lymphocyte proliferation and accumulation of ribonucleic acid per cell may prove to be a more sensitive assay of histocompatibility than tests based only on cell proliferation (tritiated thymidine incorporation).

Rapid multiparameter analysis of cell stimulation in mixed lymphocyte culture reactions.

SUMMARY

The size-distribution of normal human lymphocytes growing in tissue culture was measured with a Quantimet 720 image-analysing computer. The proportion of cells undergoing volumetric growth after phytohaemagglutinin (PHA) stimulation was derived from a simple mathematical model describing the growth patterns of the cells up to the first mitotic division. The approach may have general application in biology as a method for measuring the proportion of growing cells by microscopy.

Measurement by Quantimet 720 of the proportion of actively growing cells in tissue cultures of human lymphocytes

Mammalian cells invariably contain a vast array of glycosylated moieties, both inside the cell and on the cell surface. There is an increasing awareness of the utility of these carbohydrates in delineating the phenotype or function of many populations of cells. To this end lectins are extremely useful reagents. Lectins are carbohydrate-binding proteins and glycoproteins of non-immune origin derived from numerous plants and animals. A wide variety of lectins with many distinct carbohydrate specificities have been isolated. Historically the most common laboratory techniques utilizing lectins have been agglutination, mitogen stimulation, and fluorescence techniques. Recent advances in the development and conjugation procedures for labels and matrices have led to the creation of numerous novel lectin-based assays. Lectins are currently used not only to identify cells with specified carbohydrate groups, but also to quantitate the carbohydrate groups or to isolate the carbohydrate-bearing cells or structures

The application of lectins to the characterization and isolation of mammalian cell populations.

Quantitation of lymphocyte proliferative response to allogeneic cells and phytohemagglutinin by flow cytofluorometry. II. Comparison with [14C]thymidine incorporation.

T.K. Sharpless

Papers

Quantitation of lymphocyte proliferative response to allogeneic cells and phytohemagglutinin by flow cytofluorometry. II. Comparison with [14C]thymidine incorporation.