Abstract
Cytokinesis is an intensively studied process by which the cell cytoplasm divides to produce two daughter cells. Like any other aspect of cell cycle research, the study of cytokinesis relies heavily on cell synchronization. However, the synchronization of cells during cytokinesis is challenging due to the rapid nature of this process and the shortage of cell cycle blocking agents specifically targeting this phase. Here, we demonstrate the use of standard flow cytometry for directly isolating cytokinetic cells from an asynchronous population of normally proliferating cells. This approach is based on a cell cycle marker whose temporal proteolysis, in combination with DNA quantification or cell size approximation, distinguishes cells undergoing cytokinesis. Furthermore, by avoiding doublet discrimination, typically used in flow cytometry analyses, we were able to further increase selectivity, specifically purifying cells at late cytokinesis. Our method circumvents checkpoint activation, cell cycle arrest, and any other means of pre-synchronization. These qualities, as demonstrated for both unattached and adherent cells, enable high selectivity for cytokinetic cells despite their overall low abundance in an asynchronous population. The sorted cells can then be readily used for cell biological, biochemical, and genomic applications to facilitate cytokinesis and cell cycle research.
Highlights
Cytokinesis is an intensively studied process by which the cell cytoplasm divides to produce two daughter cells
We demonstrate the use of standard flow cytometry for directly isolating cytokinetic cells from an asynchronous population of normally proliferating cells
Tagged cell cycle proteins have been widely used as cell cycle markers. These fusion proteins are constitutively expressed, and their level is regulated by the ubiquitin-proteasome system, such that the markers’ temporal proteolysis indicates distinct points along the cell cycle[14,15,16]
Summary
Cytokinesis is an intensively studied process by which the cell cytoplasm divides to produce two daughter cells. Our method circumvents checkpoint activation, cell cycle arrest, and any other means of pre-synchronization These qualities, as demonstrated for both unattached and adherent cells, enable high selectivity for cytokinetic cells despite their overall low abundance in an asynchronous population. Cytokinesis is a relatively short process; the fraction of cytokinetic cells within a population of normally proliferating cells is small This limitation can be overcome by cell cycle blocking agents that pause cell cycle progression at a specific point via checkpoint mechanisms. The metaphase plate cannot be formed, the mitotic checkpoint is activated, and cells are arrested with 4 N DNA and fully condensed chromosomes This synchronization approach is effective; for example, nocodazole blocks cells at pre-metaphase with nearly 100% efficiency. Cells respond differently to drugs due to i) non-genetic heterogeneity; ii) uneven cell cycle arrest resulting from the random cell cycle position of each cell before treatment; and iii) non-cell autonomous effects
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