The number of marginal zone (MZ) B cells is reduced in WASp-deficient mice. The MZ B-cell population was detected as CD21^highCD23^low by either flow cytometry (A) or by counterstaining with the MZ macrophage-specific marker MOMA-1 (B), or as IgM^highIgD^low on spleen cryostat sections (C). (A) Spleen cells from nonimmunized wild-type (WT) and WASp-deficient (WASp–) mice were stained with PE-labeled anti-CD21 and FITC-labeled anti-CD23 antibodies, thereafter analyzed by flow cytometry using a gate for lymphocytes. The CD21^highCD23^low MZ B-cell populations are encircled in the left-hand graphs. Their percentage of the total lymphocyte population is indicated. The percentage of MZ B cells from 4 WT and 4 WASp– spleens is shown in the right-hand graph. The level of significance was calculated using a 2-tailed t test and the bracket indicates P < .001. Presented results are representative of 2 similar experiments. (B) Cryostat sections from nonimmunized WT and WASp– spleens were stained with PE-labeled anti-B220 (red) and Cy5-labeled anti-CD3 (blue) antibodies to detect B and T cells, respectively. MZ macrophages were detected with MOMA-1 antibodies followed by Alexa488-labeled secondary antibodies (green). The layer of B220⁺ B cells that surrounds the MZ macrophages is indicated with white arrows. Note the reduced thickness in the WASp– spleen section. (C) Cryostat sections of WT and WASp– spleens on day 5 in the immune response to SRBCs (diluted 1:10) were stained with PE-labeled anti-IgM (red) and FITC-labeled anti-IgD (green) antibodies to detect B cells. T-cell areas were detected using Cy5-labeled anti-CD3 (blue) antibodies. The rim of IgM^highIgD^low MZ B cells, localized at the outer border of the B-cell follicle, is indicated with white arrows. Note the reduction of IgM^highIgD^low MZ B cells in the WASp– spleen section.