CycleFlow simultaneously quantifies cell-cycle phase lengths and quiescence .

Populations of stem, progenitor, or cancer cells show proliferative heterogeneity , comprising proliferating and quiescent cells. Consistent quantification of the quiescent subpopulation and progression of the proliferating cells through the individual phases of the cell cycle has not been achieved. Here, we describe CycleFlow, a method that robustly infers this comprehensive information from standard pulse-chase experiments with thymidine analogs. Inference is based on a mathematical model of the cell cycle, with realistic waiting time distributions for the G, S, and G/M phases and a long-term quiescent G state. We validate CycleFlow with an exponentially growing cancer cell line . Applying it to T cell progenitors in steady state , we uncover strong proliferative heterogeneity, with a minority of CD4CD8 T cell progenitors cycling very rapidly and then entering quiescence. CycleFlow is suitable as a routine method for quantitative cell-cycle analysis.