Impaired proliferation and activation of BMKO B cells. (A) Littermate control and BMKO mice were supplied with BrdU-containing drinking water for 3.5 d. Top left, total splenic B cells were stained with anti-BrdU and anti-B220 Abs and analyzed by flow cytometry (n = 5; **, P = 0.0021). Bottom left, gated B220⁺IgM⁺ cells were further gated on CD93⁺ and sorted to detect BrdU⁺ TS B cells (n = 4; *, P = 0.024). Top right, B220⁺IgM⁺ cells were gated on CD21⁺CD23^low and sorted to detect BrdU⁺ MZ B cells (n = 4; ***, P = 0.000077). Bottom right, B220⁺IgM⁺ cells were gated on CD21⁺CD23⁺ and sorted for BrdU⁺ FO B cells (n = 4; ***, P = 0.0001). Numbers are the percentage of BrdU⁺ cells among the gated population. (B) Purified splenic littermate control and BMKO B cells were stimulated for 24 h with the indicated doses of anti-IgM (left) or anti-IgM plus anti-CD40 (right) and incubated with [³H]-thymidine. Results are the mean ± SD of triplicate. (C) Purified splenic littermate control and BMKO B cells were stimulated with 10 µg/ml anti-IgM or 10 µg/ml anti-IgM plus 0.5 µg/ml CD40 ligand for 24 h. Up-regulation of the indicated activation markers was monitored by flow cytometry. (D) Purified splenic littermate control and BMKO B cells were stimulated with 20 µg/ml anti-IgM for the indicated times. Lysates were immunoblotted to detect phospho-tyrosine (pTyr). (E) Splenic littermate control and BMKO B cells preloaded with Indo-1 were stimulated first with anti-IgM and then with thapsigargin (TG; positive control). The Ca²⁺ response was measured based on the ratio of violet (Ca²⁺ bound) to blue (Ca²⁺ free) fluorescence. Results (A–E) are representative of two to three independent experiments involving one to five mice per genotype.