COMPARATIVE FLOW CYTOMETRIC ANALYSIS OF DNA-BOUND PCNA AND DNA CONTENT AS ESTIMATORS OF S-PHASE CELLS IN CELL-CULTURES

Flow cytometric estimations of S-phase cells were carried out on cultu res from three different cell lines and in frozen aliquots. A PCNA-ext raction protocol was applied. Measurements of the S fraction estimated from bivariate PCNA/DNA analysis after detergent extraction of DNA no n-bound PCNA were compared with those obtained from total DNA histogra ms (Vindelov and Christensen's technique, methanol-fixed whole cells a nd PCNA-extracted nuclei), No significant differences between methods, or between fresh and frozen specimens, were found in the measurements of the percentage of S-phase cells. Nevertheless, nuclei yield follow ing PCNA extraction was highly variable, ranging from 63% to 10% (mean : 26%). In some cases, the extraction was not complete and samples had to be discarded. Usually, boundaries between S-phase events and G0/G1 or G2/M subpopulations were not clearly defined, Because of these sho rtcomings, and the fact that is more costly and time consuming, the es timation of the S-phase fraction by means of bivariate DNA-bound PCNA/ total DNA flow cytometric studies does not seem to surpass that obtain ed from standard DNA cell cycle analyses.