Reversible synchronization of cultured rodent and human diploid fibroblast cells in G2 phase

This paper describes a three-step procedure for induction of reversible cell synchrony in the G₂ phase of the cell cycle of Chinese hamster ovary (line CHO) cells and non-transformed, human skin fibroblast (HSF) cells. The CHO cells are presynchronized in early S phase by isoleucine deficiency and hydroxyurea blockades. The culture is transferred to medium supplemented with the DNA topoisomerase II inhibitor, Hoechst 33342 for 12 hours after which 95% of the cells are arrested in G₂ phase. When G₂ synchronized cells are transferred to Hoechst-free, complete medium, they divide as a highly synchronous cohort within 3 hours. The HSF cells are initially cultured in low serum to arrest them in G₀ and then transferred to complete medium containing aphidicolin to arrest them in early S phase. The culture is then transferred to aphidicolin-free, complete medium with Hoechst 33342 (0.1 g/ml) for 10 hours after which up to 85% of the cells arrest in G₂ phase. Synchronous cell division occurs 3.5 hours after transfer of cells to complete, drug-free medium. The synchrony techniques are useful for studying G₂/M biochemical events in mammalian cells.Abbreviations -MEM minimum essential medium alpha medium - BCS bovine calf serum - DMSO dimethyl sulfoxide - EDTA ethylenediaminetetraacetic acid - FCM flow cytometry - PBS phosphate buffered saline - Top II DNA topoisomerase II