The effect of TLR9 signaling on the outcome of B cell responses to antigen In all cases purified mouse splenic B cells (WT or TRL9 KO) were stimulated in vitro with Anti-IgM (2–5μg/ml) or CpG (1μM) alone or in combination. (a–d) , pooled, permeabilized and stained with mAbs specific for the phospho-kinases: p-Syk (a) , p-Btk (b) , p-p38 (c) and p-Akt (d) . The fold changes in abundance of phosphorylated kinases in stimulated as compared to unstimulated B cells are shown. (e) Calcium flux measured by flow cytometry in B cells loaded with the Ca 2+ sensor dyes Furo-red and Fluo-4 and stimulated. (f) Fold changes in the mRNA expression for various cytokines of B cells stimulated for 4h as compared to unstimulated B cells. ( g) ELISA measurements of cytokine proteins in the culture supernatants of WT or TLR9 KO B cells stimulated in vitro for 18 h (for IL-6) or 24 h (for TNF, IL-2 and IL-10). (h) Proliferation of WT or TLR9 KO B cells stimulated with a sub-optimal concentration of Anti-IgM (1μg/ml) and increasing concentrations of CpG (0 to 3 μM). Shown are the percentage of cells that proliferated after 46 h of culture. (i,j) Antibody production by stimulated B cells for a duration of seven days. ELISA measurement of IgM (i) and IgG from the IgG + ) (j). (k–m) Kinetic analysis of in vitro mRNA expression of GC B cell- or PC-specific genes in stimulated WT B cells for 4 days. Expression of Bcl6 ( k ), Prdm1 ( l ) and Aicda ( m ) is shown as fold changes over that observed in unstimulated B cells at time 0. Data are representative of three independent experiments performed with duplicate ( a–d ), or triplicate samples ( e–n) . Data points and error bars indicate mean and standard deviation, respectively. Statistical significance was measured using two sided unpaired t-test (**= 0.001