Flow cytometry after bromodeoxyuridine labeling to measure S and G2+M phase durations plus doubling times in vitro and in vivo

Abstract

This protocol describes methods for calculating the proliferative parameters of cell populations. The basis of the technique is to label cells, either in vitro or in vivo, with halogenated thymidine analogs, such as bromodeoxyuridine (BrdU). Bivariate DNA-BrdU flow cytometry is used to analyze the BrdU-labeled and unlabeled cells. The enumeration of specific cohorts of cells that either have or have not divided in the interval between labeling and cell/tissue sampling permits the calculation of the potential doubling time (T(pot)) of the population, plus the durations of DNA synthesis (T(S)) and the G2+M phase (T(G2+M)) of the cell cycle. The method provides information that is not otherwise available, namely inhibition of DNA synthesis and the separate evaluation of cell-cycle effects in BrdU-labeled and unlabeled subpopulations. Ethanol-fixed samples take 1 d to prepare and stain, and reliable parameter estimates might be obtained from measurements made at a single time point after labeling.