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Z Darzynkiewicz

Publications and source records attributed to Z Darzynkiewicz.

28 records · Page 2Linked to original sources
PubMed1977

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

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.

Acridines↗
PubMed1977

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

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).

Acridines↗
PubMed1977

Recognition of cells in mitosis by flow cytofluormetry.

Cells in mitosis may be distinguished from interphase cells based on difference in chromatin structure as revealed by two different methods of staining with acridine orange. In the first method, cells are heated and then stained at neutral pH; the difference in stainability between mitotic and interphase cells reflects the difference in the extent of deoxyribonucleic acid denatured by heat in these cells. At a given temperature the deoxyribonucleic acid of the mitotic cell appears to be more extensively denatured than that of the interphase cell. In the second method, cells are treated with buffer at pH 1.5 (1.3 to 1.9) and then stained at pH 2.6 (2.3 to 2.9). The mechanisms involved in the differential stainability of interphase versus mitotic cells at that low pH are currently under investigation. In both methods, in addition to enumerating cells in mitosis, it is possible to quantitate cells in G1, S and G2 phases of the cell cycle.

Acridines↗
PubMed1977

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.

Cell Cycle↗
PubMed1977

Nucleic acid content and nuclear chromatin structure of human bladder cell culture lines as studied by flow cytofluorometry.

Two human bladder cell lines, T-24 and HCV-29, are studied with flow cytofluorometry and acridine orange staining to determine relative DNA and RNA content per cell and to measure resistance to thermal denaturation of DNA in situ. The RNA/DNA ratio for HCV-29 is over twofold higher than that for T-24, a difference that is consistent with the differences in cytological morphology and staining characteristics of these two cell lines and is sufficient to distinguish them completely, although measurements of DNA or RNA alone may not. In addition, the two cell lines show differences in DNA "melting" curves that indicate structural or conformational differences in nuclear chromatin. It is evident that the features are related to nuclear and cellular morphology, and they may be of value as additional parameters for characterizing tissue culture cell lines.

Cell Line↗
PubMed1976

Lymphocyte stimulation: a rapid multiparameter analysis.

Several parameters of stimulation of individual lymphocytes are measured simultaneously by flow-cytofluorometry after differential staining of cellular DNA and RNA with the metachromatic fluorescent dye acridine orange. The method provides a means of analyzing the progression of stimulated cells through the cell cycle (G1, S and G2 + M), in addition to measuring the accumulation of RNA per cell; the RNA parameter is useful in distinguishing G1 from G0 cells. The multiparameter analysis may be of unique value in discriminating cases where the transcriptional and proliferative responses of lymphocytes to stimulants are not correlated.

Acridines↗