Impaired BCR signaling and reduced proliferation of DKO B cells to both anti-IgM and LPS.

<p>A. Intracellular calcium response to anti-IgM using control and mutant B cells. <i>Gnai2</i>^fl/fl, <i>Gnai2 </i>^fl/fl<i>mb1-cre</i>, Gnai3^-/-, and DKO B cells were stimulated with anti-IgM and the induced changes in intracellular calcium were monitored over 3 minutes, left panel. The data shown as fluorescent counts and the y-axis labeled as iLm1. Each experimental value is the mean of three determinations. Similar experiment comparing the intracellular calcium responses of B cells prepared from <i>Gnai2</i>^fl/fl or <i>Gnai2 </i>^fl/fl<i>mb1-cre</i> mice to increasing concentrations of anti-IgM, right panel. Experiments repeated three times with similar results. B. Flow cytometry results from the analysis of B220, IgD, and IgM expression on spleen B cells from <i>Gnai2</i>^fl/fl, <i>Gnai2 </i>^fl/fl<i>mb1-cre</i>, Gnai3^-/-, and DKO B cells. For each genotype B220 expression is shown on cells in the lymphocyte gate and a second plot of IgM versus IgD expression after gating on B220⁺ cells. Data is representative of multiple experiments. C. Dye dilution assay to assess the initial proliferative potential of wild type and mutant B cells. B cells from control and mutant strains were stained with Pacific blue and then cultured with the indicated stimulants for 96 h. The percentage of cells that divided in each cultured was determined by the analysis of flow cytometry data. The results are from the analysis of B cells from two mice of each genotype. Statistics are from analysis of the data by 2 way ANOVA. ** p<0.001, * p<0.05.