New methods of cell cycle analysis based on the selective tagging of cells either at the beginning or end of S phase.

Abstract

New techniques for cell cycle analysis are presented. Using HeLa cells, methods are described for the selection of a narrow window or cohort of lightly [3H]-labeled cells located either at the very beginning or the very end of S phase. The cohort cells are tagged by a labeling procedure which entails alternating pulses of high and low levels of [3H]thymidine and are identified autoradiographically. Additional methods are described for following the progress of cohort cells through the cell cycle. Theoretically, with the methods described, it should be possible to follow the "early S cohort' cells as they exit from S phase, as they enter and exit M and as they enter the subsequent S phase. This would allow a determination of S, S + G2, S + G2 + M and T. It should theoretically be possible to follow "late S cohort' cells in a similar manner, allowing a determination of G2, G2 + M and G2 + M + G1. To test these predictions, several experiments are presented in which the progress of the two cohorts is monitored. The best data were obtained from the mitotic curves of cohort cells. For each of the cohorts, values were obtained for the time required for peak concentration of cells in mitosis, the coefficients of variation and of skew. The curve of cohort cells passing through mitosis is shown to fit a log-normal curve better than a normal curve. In addition, the mitotic curves are used to estimate the length of M and to estimate the loss of cohort synchrony. Other uses of these methods are discussed.