Application of BrdU/Hoechst-ethidium bromide two parameter flow cytometry in studying synchronous and non-synchronous...

BrdU/Hoechst-EB bivariate flow cytometry has a wide application in the study of factors controlling cell cycle for asynchronous cells such as embryonic stem cells (ES), and for synchronous cells such as stimulated resting B cells (Bo). The technique allows one to calculate the average cell cycle duration time. ES cells are found to cycle every 8-10 h, and most B cells are 11-12 h, but there is a small subset of B cells with a cycle time of only 6-7 h. Using this technique, we also study the roles of different T lymphocytes on B cell activation when B cells are stimulated with anti-IgM antibodies (commonly used, anti-mu). Exposure to anti-mu recruits small B cells into the cell cycle, but arrests them in the G1 phase of the second cycle. Interleukin (IL) 4 is a costimulator of anti-mu. In addition, IL-4 is an S-phase progression factor. Contrary to that seen when B cells are stimulated by other mitogens, very few cells are in the G2 compartments after anti-mu plus IL-4 stimulation. This phenomenon is reminiscent of embryonic cells. Our findings provide strong evidence to propose that there are two restriction points for B cell activation: at the transition from G0 to G1 and at the transition from G1 to S phase.