New cell cycle compartments identified by multiparameter flow cytometry

Abstract Simultaneous measurements of cellular DNA and RNA as well as estimates of the sensitivity of DNA in situ to denaturation by acid (which correlates with the degree of chromatin condensation) and cell ability to incorporate 5‐bromodeoxyuridine (BUdR), performed on over 40 different cell systems enabled us to subclassify cells into 12 functionally distinct cell cycle compartments. Quiescent cells had low RNA values, DNA very sensitive to denaturation, and they did not incorporate BUdR. Although in most cell systems they had 2C DNA content (G 1Q ), quiescent cells with higher DNA values (S Q and G 2Q ) were also seen. The G 1 phase of exponentially growing cells had two distinct compartments, A and B. G 1 cells entered S phase only from the B compartment. An increase in RNA and a decrease in the sensitivity of DNA to acid denaturation beyond a specific level characterized the transition of cells from the A to B compartments. Since the G 1A to G 1B transition was not linear but exponential, it may be assumed that a non‐deterministic event triggering cell progression into the cycle resides within G 1A . Under adverse growth conditions the probability of a G 1A to G 1B transition decreased. Cells In G 2 had DNA more sensitive to acid denaturation than S or G 1 phase cells. Mitotic cells had the most condensed chromatin and their RNA content was twice that of G 1A cells. Differentiated cells were characterized by 2C DNA (G 1D ) but, depending on the cell type, had varying RNA content and different degrees of chromatin condensation. A hitherto undescribed category of cells undergoing transition from quiescence to the cycle (or vice versa) was distinguished based on their intermediate values of RNA, sensitivity of DNA to acid denaturation and inability to incorporate BUdR at the rate characteristic for S cells. Depending on their DNA content (C) at the time of transition these cells could be classified as G 1T (2C), S T (4>C>2) or G 2T (4C).