Flow cytofluorometric analysis of cell cycle distributions using propidium iodide. Properties of the method and mathematical analysis of the data
Flow cytofluorometric analysis of cell cycle distributions using propidium iodide. Properties of the method and mathematical analysis of the data
复制标题
使用碘化丙啶对细胞周期分布进行流式细胞荧光分析。
作者：
J. Fried;A. G. Pérez;B. Clarkson
为了更好地表征克里山提出的新的流式细胞荧光快速染色方法，我们对其稳定性和其他几个性质进行了测试，并对用碘化丙啶和吖黄素-福尔根染色方法得到的荧光直方图进行了定量比较。以处于对数生长期的人造血细胞系为材料，用我们设计的数学方法对数据进行分析，发现碘化丙啶染色的细胞在至少48h内荧光强度保持稳定，在0.025-0.10mgml(37-150um)范围内对染料不敏感，染色前与核糖核酸酶孵育不影响，0.1%柠檬酸钠溶液中的碘化丙啶至少稳定20d；对细胞周期不同时相细胞所占比例的定量估计与吖黄素-Feulgen染色和氚胸苷脉冲标记后放射自显影的结果一致。我们的结论是，这种方法对于用流式细胞荧光法测量相对DNA含量是有用的，尽管对于生长在单层中的某些细胞类型来说，技术上的改进是必要的。
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 intentsity 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 muM); 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.