B Cell Stimulation and Isolation for Microarray Analysis

B cells purified from splenocytes by positive selection using anti-B220 antibody beads (Miltenyi Biotec), were cultured in the complete medium (RPMI 1640 containing 10% FBS, 1% penicillin/streptomycin and 0.1% 2-mercaptoethanol (ME), all from Life Technologies) at the density of 2 × 10⁶ cells/ml. Cells were stimulated with anti-CD40 antibody (2 μg/ml, clone HM40-3, BD Pharmingen) or LPS (10 μg/ml) for 5–48 h. In some experiments, phorbol-12-myristate-13-acetate (PMA, 50 ng/ml, BD Pharmingen), ionomycin (500 ng/ml, Sigma-Aldrich), and monensin (2 μM, eBioscience) were added in culture for the final 5 h before the detection of intracellular IL-10 (29 (link)). In microarray analysis, splenic B cells were treated with either LPS or anti-CD40 for 48 h, and IL-10⁺ or IL-10⁻ B cells were then purified by Regulatory B Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer's instructions. To compare the genes differentially expressed in Ctrl and Cko B10 cells, isolated B10 cells from Ctrl and Cko mice were stimulated with anti-CD40. RNA samples were collected at 0 and 48 h after stimulation.