An improved mathematical method to estimate DNA synthesis time of bromodeoxyuridine‐labelled cells, using FCM‐derived data

Abstract

Abstract. Chinese hamster ovary cells in vitro were pulse‐labelled with bromodeoxyuridine (BrdUrd and were then allowed to progress through the cell cycle. Every half hour after labelling, cells were harvested and prepared for simultaneous flow cytometric determination of DNA content and incorporated BrdUrd, with the intercalating dye propidium iodide and with a monoclonal antibody against incorporated BrdUrd, respectively. The relative movement (RM), i.e. the relative mean DNA content of the moving cohort of BrdUrd‐labelled cells in relation to that of G1 and G2 cells, was calculated. RM was then used to calculate DNA synthesis time (TS), at all post‐labelling times (t). Since labelled cells in G2 and mitosis (M) in addition to S phase cells, are included in the cohort of moving labelled cells, and since the time of G2 and M (Tg2+M) phases is finite, a non‐linear relationship exists between RM and post‐labelling time. Because of this, the use of a linear formula in the calculation of TS yields results that are affected by t. We found that RM data can be corrected with regard to TG2+M resulting in the derivation of a non‐linear TS formula. This non‐linear TS formula gave results that were nearly independent of t. Moreover, windows were set in the mid DNA distributions for G1, S and G2+ M cells in the bivariate DNA v. BrdUrd cytograms, to estimate the fraction of BrdUrd‐labelled cells in each window at every post‐labelling time. Plots of the fraction of BrdUrd‐labelled cells v. post‐labelling time were then made for each window. TS obtained in this way was in agreement with TS obtained with the corrected RM method. In conclusion, we present a method to calculate Ts which theoretically first makes the determination of RM independent of TG2+M, and secondly compensates for the non‐linear function of RM with post‐labelling time caused by accumulation of BrdUrd‐labelled cells in G2+ M.