Cell Synchrony Techniques. I. A Comparison of Methods

Abstract Selected cell synchrony techniques, as applied to asynchronous populations of Chinese hamster ovary (CHO) cells, have been compared. Aliquots from the same culture of exponentially growing cells were synchronized using mitotic selection, mitotic selection and hydroxyurea block, centrifugal elutriation, or an EPICS V cell sorter. Sorting of cells was achieved after staining cells with Hoechst 33258. After synchronization by the various methods the relative distribution of cells in G₁ S, or G₂+ M phases of the cell cycle was determined by flow cytometry. Fractions of synchronized cells obtained from each method were replated and allowed to progress through a second cell cycle. Mitotic selection gave rise to relatively pure and unperturbed early G₁ phase cells. While cell synchrony rapidly dispersed with time, cells progressed through the cell cycle in 12 hr. Sorting with the EPICS V on the modal G₁ peak yielded a relatively pure but heterogeneous G₁ population (i.e. early to late G₁). Again, synchrony dispersed with time, but cell-cycle progression required 14 hr. With centrifugal elutriation, several different cell populations synchronized throughout the cell cycle could be rapidly obtained with a purity comparable to mitotic selection and cell sorting. It was concluded that, either alone or in combination with blocking agents such as hydroxyurea, elutriation and mitotic selection were both excellent methods for synchronizing CHO cells. Cell sorting exhibited limitations in sample size and time required for synchronizing CHO cells. Its major advantage would be its ability to isolate cell populations unique with respect to selected cellular parameters.