Evaluation of flow cytometric methods for determining population potential doubling times using cultured cells

Various methods have been proposed for determining the potential doubling times (Tpot) of mammalian cell populations by using flow cytometric techniques after labeling the cells with bromodeoxyuridine (BrdUrd). We show here that, in a well-defined in vitro system where multiple time measurements are possible, all the methods give similar results that are close to the true population doubling time. Of ultimate interest, however, is the accuracy of determination of Tpot from a single time point. In this paper we compare the accuracy and precision of the methods in making such determinations at different times after labeling. The relative movement (RM) of BrdUrd-labeled cells that have not divided at the time of assay allows for computation of the length of S phase (Ts). The precision of estimation of Ts was enhanced when a quantity, v (a function of the fraction of BrdUrd-labeled divided and the fraction of BrdUrd-labeled undivided cells), was used to estimate the initial intercept of RM. Furthermore, calculation of Tpot from the formula, Tpot = ln(2) Ts/v, gave values closest to the observed population doubling time. It is suggested that the use of RM with v be the analytical method of choice for the calculation of Tpot from single time-point observations, preferably made at times between the length of the G2 and M phases (TG2M) and Ts.