Application of BrdU/Hoechst-ethidium bromide two parameter flow cytometry in studying synchronous and non-synchronous mouse cells.
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.
D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000494:pharmacology; D001402:B-Lymphocytes / Q000737:chemistry / Q000166:cytology* / Q000276:immunology; D006690:Bisbenzimidazole; D001973:Bromodeoxyuridine; D002453:Cell Cycle / Q000187:drug effects / Q000276:immunology; D002455:Cell Division; D004996:Ethidium; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D006131:Growth Inhibitors / Q000494:pharmacology; D007075:Immunoglobulin M / Q000276:immunology / Q000494:pharmacology; D015847:Interleukin-4 / Q000494:pharmacology; D015850:Interleukin-6; D007399:Interphase / Q000187:drug effects / Q000276:immunology; D053605:Leukemia Inhibitory Factor; D008213:Lymphocyte Activation / Q000187:drug effects; D008222:Lymphokines / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008819:Mice, Nude; D013234:Stem Cells / Q000737:chemistry / Q000166:cytology / Q000276:immunology
D000276:Adjuvants, Immunologic; D000888:Antibodies, Anti-Idiotypic; D006131:Growth Inhibitors; D007075:Immunoglobulin M; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; C507838:Lif protein, mouse; D008222:Lymphokines; C044203:anti-IgM; D015847:Interleukin-4; D004996:Ethidium; D001973:Bromodeoxyuridine; D006690:Bisbenzimidazole