Fig 1: Normal splenic B-cell development and function upon hematopoietic deletion of the miR-497/195 cluster. (A) Gating strategy for the identification of different B-cell subsets in the spleen. CD19+B220+ B2 B cells were divided into mature (AA4.1-) and immature B cells (AA4.1+). The mature B cells were further split into CD1d+ marginal zone B cells and CD1d- follicular B cells. Immature B cells were gated for the different transitional phases T1 (IgM+CD23-), T2 (IgM+CD23+), and T3 (IgM-CD23+). (B) Bar graphs indicate the mean percentages of the indicated B-cell population of miR-497/195+/+ Vav-Cre control (n = 7) and miR-497/195fl/fl Vav-Cre mice (n = 8) within the B2, mature, or immature B-cell pool. Each dot represents the data derived from one mouse. (C, D) Splenocytes of control (n = 5) or miR-497/195fl/fl Vav-Cre mice (n = 6) were labeled with a proliferation dye and stimulated with either anti-IgM or anti-CD40 antibodies together with IL-4 for 72 h. The percentage of proliferated B cells (B220+) was quantified by flow cytometric analysis, and the proliferation index was calculated as the total number of divisions normalized to the number of divided cells (C). (E) Serum immunoglobulin levels for IgM, IgG1, and IgA were measured by ELISA and calculated according to the standard curve. The graph for IgG3 depicts the optical density (OD) as measured by the plate reader (n = 6). (F, G) For immunizations, at least six control and miR-497/195fl/fl Vav-Cre mice were injected with TNP-Ficoll (F) or with NP-CGG (G) at d0. TNP-specific IgM or IgG3 levels as well as NP-specific IgM or IgG1 levels were quantified at the indicated time points by ELISA. Error bars depict the standard deviation of the mean.