Figure 4. Btk regulates peritoneal B-1 cell survival.

A) The frequency of liposome-binding cells among all peritoneal cells is indicated. Data are mean +/- SD, n = 3 for 6-1, 4 for 6-1.Btk^lo, and 2 for 6-1.Btk-/-. B) Peritoneal cells were stained with FITC-encapsulated liposomes, anti-CD5 PE, anti-B220 PerCP and anti-CD23 biotin + streptavidin APC. Liposome-binding (top) or B220+ (bottom) cells within the lymphoid gate were examined for CD23 and CD5 expression. Data are representative of 3 mice for 6-1 and 4 for 6-1.Btk^lo. C) B220+ cells were purified from the peritoneum of pools of 2-3 mice per group using magnetic beads or flow cytometry. Cells were plated in 96 well plates at 10⁶ per well. The number of live cells (as measured by trypan blue exclusion) remaining at the indicated time is indicated. Data represent the mean +/- SEM, n = 6 for 6-1 and 5 for 6-1.Btk^lo. D) B220+ cells were purified from the peritoneum of pools of 2-3 mice per group using magnetic beads. IL-10 mRNA levels were measured by quantitative real-time PCR. GAPDH was used as a loading control. The relative expression of IL-10 in 6-1.Btk^lo cells compared to 6-1 wt cells (100%) is shown as mean +/- SD, n=3. E) Peritoneal cells were cultured at 2 × 10⁶ per ml for 3 days. IL-10 levels in the culture supernatants were measured by ELISA. Each symbol represents the average of duplicate samples from an individual mouse (n = 3).