Studies on the division cycle of mammalian cells III. Preparation of synchronously dividing cell populations by isotonic sucrose gradient centrifugation

Abstract

A method of preparing synchronous cell cultures is presented which is based on a selection of postmitotic cells by density gradient centrifugation. Gradients were prepared by appropriate mixing of normal culture medium with medium in which NaCl was replaced by sucrose at a concentration providing for isotonicity. The cells sedimenting most slowly (2–5% of the original cell number) were collected, suspended in sucrose-free medium and cultured under conditions of an optimal steady state. Cell viability, as determined by the capacity of cells to form macroscopically visible colonies in a semisolid medium, was not altered by this treatment. Rhythmic variations of mitotic activity were observed, with a first maximum at 8 h after collection of postmitotic cells. The percentage of cells in the S period was determined at regular time intervals by pulse labeling with 3H-thymidine. A high proportion of DNA-synthesizing cells was found during low mitotic activity, while a low proportion of cells in S phase was observed at the time of maximum mitotic activity.