An ultra-pure in vitro phase synchrony method employing centrifugal elutriation and viable flow cytometric cell sorting.
We present a method of synchronizing cells in G1-, S-, and G2M-phases employing sequential centrifugal elutriation and viable flow cytometric cell sorting of Hoechst-33342 stained Chinese hamster ovary cells. G1- and S-phase cells can be separated to greater than 99% homogeneity and G2-M to 70% purity. Most of the 30% contamination in the G2-M fraction was due to S-phase cells, whose reproductive integrity could be eliminated through the use of high specific activity 3H-TdR. There were minimal toxic effects or perturbations to growth following the selection procedures. The most significant limitation of this technique appears to be the rate of cell sorting, which, with current equipment, is approximately 3,000 cells per second.
D000818:Animals; D002455:Cell Division; D002460:Cell Line; D002498:Centrifugation / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D010053:Ovary