Centroblasts with low MHCII are preferentially in G1 and early S phases of cell cycle but are underrepresented in populations that have completed fewer recent mitotic events. (a) Mice were immunized with NP-CGG/alum and then given a single i.p. BrdU treatment on day 9. Spleens were harvested at various time points, and CXCR4^high MHCII^low and CXCR4^high MHCII^high CD95⁺ GL7⁺ GC B cells were gated. (b) BrdU incorporation and DNA content were assessed at 30 min after BrdU treatment to determine cell cycle status. (c) The proportion of cells in G2/M (4n, BrdU⁻) was determined for multiple mice. (d) The proportion of BrdU⁺ Dapi^low (G1) cells was determined at the different time points. (e) NP-CGG/alum–immunized mice received treatments of EdU and BrdU by single i.p. injections 8 h (EdU) and 30 min (BrdU) before euthanasia. The respective IgD^low CD95⁺ GL7⁺ GC B cell populations were gated. Control mice, shown on top, received either EdU or BrdU alone. (f) Summarized data from multiple mice analyzed as in e. (g–l) Tet-off H2b-GFP mice were immunized with NP-CGG/alum and were subsequently treated with doxycycline (or saline) for the final 40 h before analysis on day 9. (g) GFP fluorescence was assessed in naive and GC B cells. WT nonfluorescent cells are shown as shaded areas. GFP^dim and GFP^bright, GC B cells (IgD^low CD95⁺ GL7⁺) from dox-treated mice were gated as shown in h, and the frequency of the different subsets was examined (i). Data from multiple experiments and mice are summarized in j. (k) The frequency of CXCR4^low cells within the GFP^dim and GFP^bright gates was calculated. (l) A similar analysis to j was performed but pregating only on CXCR4^high cells. Data in a–d are from one representative experiment of three, with each experiment containing one to three mice per time point. Means are shown in d (±SD). Data in e and f are from one representative experiment of two, with each experiment containing three double-labeled mice. Data in g–l are pooled from three experiments each containing two mice per condition. Each circle in c, f, and k represents individual mice. All FACS plots are from representative mice for the respective experiments. Analysis was performed using an unpaired two-tailed Student’s t test. Horizontal lines in c, f, and j–l indicate means. **, P < 0.01; ***, P < 0.001.