Flow cytofluorometric analysis of cell cycle distributions using propidium iodide. Properties of the method and mathematical analysis of the data.

Abstract

In order to better characterize the new rapid staining method for flow cytofluorometry proposed by Krishan, we have tested its stability and several other properties, and have carried out a quantitative comparison of the fluorescence histograms obtained using propidium iodide or the acriflavine-Feulgen staining procedure.Using a human hematopoietic cell line in the logarithmic phase of growth, and analyzing the data by means of a mathematical method we have devised, we found that the fluorescence intensity of cells stained with propidium iodide remains stable for at least 48 h; it is insensitive to dye concentration between 0.025 and 0.10 mg/ml (37-150 tzM); it is not affected by incubation with ribonuclease before staining; propidium iodide in 0.1% sodium citrate remains stable for at least 20 days; and quantitative estimates of the fractions of cells in the different phases of the cell cycle are in good agreement with those obtained from acriflavine-Feulgen staining and from autoradiography after pulse labeling with tritiated thymidine. We conclude that this method is useful for the measurement of relative DNA content by flow cytofluorometry, although modifications in the technique are necessary for some cell types which grow in monolayers.Because it can be used to measure selected properties of large numbers of individual cells in a population rapidly and conveniently, flow cytofluorometry has become increasingly popular as a research tool, and its potential for clinical application is being intensively explored. In principle, the intercellular distribution of any cellular component can be measured provided an appropriate fluorescent dye can be used which binds stoichiometrically to the component in sufficient quantity to be detectable by the available instrumentation. Most applications of the method have until now been concerned with the determination of the relative distribution of DNA content among the cells of a population. A method that permits one to carry out rapid determinations of relative DNA content has important implications not only as a more convenient and potentially more accurate means of cell cycle analysis of populations in culture, but also for monitoring the status and kinetic response to therapy of patients with leukemia or other neoplasms (8,9,12,13).Until very recently, the fluorescent staining methods used by most investigators were the acri-

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Interaction of canine distemper virus nucleocapsid variants with 70K heat-shock proteins

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