Purifying Cytokinetic Cells from an Asynchronous Population

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.