A comparison of the use of bromodeoxyuridine and [3H]thymidine in studies of the cell cycle.

Abstract

Untransformed Syrian hamster fibroblasts in short-term culture were studied by means of [3H]thymidine autoradiography combined with differential staining after continuous exposure to bromodeoxyuridine. Fractions of 3H-labelled metaphases (FLM) and of differentially stained metaphases (FDM) were determined in sequential samples. The resulting FLM and FDM curves are in close agreement, in sequential samples. The resulting FLM and FDM curves are in close agreement, except at later sample times (10 hr onwards), when the FLM is greater than the FDM. This inefficiency on the part of the bromodeoxyuridine technique in the detection of cells in early S-phase leads to an underestimate of the average duration of S compared with that derived from autoradiography. Grain counts suggest that only a small proportion of total DNA synthesis is missed. FLM and FDM curves from both diploid and tetraploid cells are very similar. Despite the inefficiency in the detection of cells in early S phase, the bromodeoxyuridine technique has a number of useful features in studies of the cell cycle. It is possible to distinguish pre-S cells from G2 cells unambiguously, and cells in their first cycle can be distinguished from those in their second with a much greater efficiency than by autoradiography. The technique is consistent and has no problems from background or the consequent need for thresholds in scoring. As is demonstrated here, the two methods can be combined, the differential staining being little affected by the autoradiographic procedures.