Flow cytometry to sort mammalian cells in cytokinesis.
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.


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.
Gasnereau
Isabelle
I
Université Pierre et Marie Curie - Paris 6, CNRS UMR 7098, Paris F-75005, France.
Ganier
Olivier
O
Bourgain
Florence
F
de Gramont
Armand
A
Gendron
Marie-Claude
MC
Sobczak-Thépot
Joëlle
J
D064127:Aurora Kinases; D002469:Cell Separation / Q000379:methods*; D019926:Cyclin B / Q000096:biosynthesis; D056744:Cyclin B1; D048749:Cytokinesis / Q000502:physiology*; D005434:Flow Cytometry / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis / Q000235:genetics / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis
C534699:CCNB1 protein, human; D019926:Cyclin B; D056744:Cyclin B1; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases