Mitotic cell analysis by means of univariate flowстатья
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Статья опубликована в журнале из перечня ВАК
Статья опубликована в журнале из списка Web of Science и/или Scopus
Дата последнего поиска статьи во внешних источниках: 7 сентября 2017 г.
Аннотация:A new method of mitotic cell analysis is suggested. The method is based on the univariate flow cytometry and provides a possibility of not only mitotic cells counting but also of discrimination between metaphase (Mt) and anaphase (Ma) cells. Pretreatment of cells includes disruption of cell membranes and thus releases cell nuclei from G1, S, G2 cells and chromosomes from mitotic cells. High resolution flow analysis of the DNA content both in cell nuclei and in chromosomes + chromatids appearing from mitotic cells is achieved by parallel fluorescence signal acquistion with two different gains: lower gain for cell signals and 16-times higher gain for chromosomal signals. For analysis of data two experimental univariate distribution are join in one histogram, and the "chromosomal" part of the histogram undergoes deconvolution into three components: chromosomes, chromatids, and debris. The integration of the DNA content in different components of the joint histogram gives the relative numbers of cells in different subpopulations: G1, S, G2, Mt, and Ma. G1, S, and G2 are obtained from the "cellular" part of the joint histogram, and Mt and Ma from the "chromosomal" component. Sensitivity of the proposed method makes it possible to detect low amounts of mitotic cells (similar to1%). The analysis of several thousands cells takes only 1-2 minutes and gives reliable statistics even in the case of a low percent of mitosis. This provides the possibility of metaphase and anaphase cell counting both in the case of unperturbed cell growth and in the cases when cell proliferation have been modified by various factors.
The approach is illustrated by experimental analysis of cell line GM 130 arrested in mitosis with nocodazoe and then washed from the blocking agent. The completion of mitosis was also studied in the presence of topoisomerase II inhibitor AMSA.