Analysis of the Length of S‐Phase Required to Show Sister Chromatid Differential Staining

Abstract

In uitro studies of BrdU-dependent sister chromatid differential staining typically employ two cycles of BrdU incorporation. Experiments are described which determined the actual fraction of both S-phases that the rat embryonic fibroblasts (Rat-1) cells had to traverse in order to show distinctive differential staining. Following synchronization of cells by a combination of serum deprivation and hydroxyurea blockage, sister chromatid differential staining, labelling index, mitotic index, and per cent DNA replication are determined. Results indicate that only -50% of the first S-phase is necessary in order to show distinctive differential staining. The importance of this finding to studies of cellular proliferation using BrdU incorporation is discussed.Recent studies of cellular proliferation have utilized 5-bromodeoxyuridine (BrdU) incorporation followed by sister chromatid differential staining (Tice, Schneider ). This system is based on culturing cells in the presence of BrdU and the subsequent identification of metaphases which have replicated for one, two or three cell cycles. It is known that exposure to BrdU for one S-phase (followed by a second S with or without BrdU) allows differential staining of sister chromatids. Most in vitro studies, however, utilize continuous BrdU labelling for both cell cycles (rather than washing out BrdU for the second cycle). In this report, we describe experiments which are designed to determine the actual fraction of both S-phases rat embryonic fibroblast (Rat-1) cells must traverse in order to show distinctive differential staining. M A T E R I A L S A N D M E T H O D S Cell cycle blockadeRat embryo fibroblast (Rat-1) cells were cultured at 39OC in McCoy's 5a medium supplemented with 10% fetal bovine serum and 1 % penicillin-streptomycin. Under these conditions, Rat-I cells were determined to have the following cell cycle parameters: generation time