A quantitative method for evaluating bivariate flow cytometric data obtained using monoclonal antibodies to bromodeoxyuridine

A method is presented for analyzing data from bivariate analysis of cell populations exposed to bromodeoxyuridine and subsequently examined both for the presence of BrdUrd and for the cellular DNA content. It is shown that certain features may be defined in the bivariate data which are constant independent both of cell type and, within limits, experimental variability. These landmark features include the ratio of red, DNA, fluorescence of G2 + M cells to G1 cells, the ratio of green fluorescence corresponding to the non-specific binding of unlabeled G2 + M cells to unlabeled G1 cells, and the distribution of green fluorescence in unlabeled cells. The landmarks make it possible to standardize rules for establishing the separation line between-labeled and unlabeled cells as required in these experiments to obtain estimates of cytokinetic parameters. Values obtained for the DNA synthesis time and the potential doubling time which result from different decision rules for distinguishing labeled from unlabeled are compared in two murine tumor lines. The potential doubling time, but not the DNA synthesis time is shown to depend sensitively on the separation line. Suggestions are presented for analyzing clinical data with this procedure.