Flow cytometry to sort mammalian cells in cytokinesis.

ABSTRACT

BACKGROUND: Cell division or cytokinesis, which results from a series of events starting in metaphase, is the mechanism by which the mother cell cytoplasm is divided between the two daughter cells. Hence it is the final step of the cell division cycle. The aim of the present study was to demonstrate that mammalian cells undergoing cytokinesis can be sorted selectively by flow cytometry. MATERIALS AND METHODS: Cultures of HeLa cells were arrested in prometaphase by nocodazole, collected by mitotic shake-off and released for 90 min into fresh medium to enrich for cells undergoing cytokinesis. After ethanol fixation and DNA staining, cells were sorted based on DNA content and DNA fluorescence signal height. RESULTS: We define a cell population that transiently accumulates when synchronized cells exit mitosis before their entry into G1. We show that this population is highly enriched in cells undergoing cytokinesis. In addition, this population of cells can be sorted and analyzed by immunofluorescence and western blotting. CONCLUSIONS: This method of cell synchronization and sorting provides a simple means to isolate and biochemically analyze cells in cytokinesis, a period of the cell cycle that has been difficult to study by cell fractionation.