Clone hm40 3

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.

Purified B cells (1 × 10⁵) were cultured in round-bottom p96 wells alone or with anti-CD40 (1 mg/ml; clone HM40-3, BD Biosciences) plus IL-4 (20 ng/ml; Peprotech) or LPS (2.5 mg/ml; Sigma). After 96 h, supernatants were collected for antibody detection by ELISA; ELISA plates were coated with goat anti-κ light chain (1 μg/ml; Southern Biotech), blocked as before, incubated with appropriate dilutions of supernatants (1:100 for IgM; 1:10 for the distinct IgG), and developed as before.

B cells were isolated from naïve mouse spleens by negative selection (Stem Cell Technologies, Vancouver, BC). The purified B cells used for all experiments were phenotypically characterized as >95% CD19⁺B220⁺IgD⁺CD23^-/lowCD5^-CD11c^-, indicating that they were predominantly B-2 marginal zone B cells [26 (link)] (S1 Fig). Purified B cells were cultured in triplicate in 96-well plates at 10⁵ cells/ well in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS; HyClone, Rockford, IL), 2% penicillin-streptomycin (Gibco, Burlington, ON, Canada), 50 mM mercaptoethanol (Gibco) and 2 mM L-glutamine (Gibco). Pam3CSK4 and poly I:C dilutions were made in complete medium and added into the wells containing B cells for a final volume of 200 μL per well. T-cell-dependent B cell activation was simulated by adding purified hamster anti-mouse CD40 (2.5 μg/mL; clone HM40-3, BD Biosciences, Mississauga, ON) and purified rat anti-mouse kappa Ig (1 μg/mL; clone 187.1, BD Biosciences) to the B cell suspensions. Plates were incubated at 37°C/ 5% CO₂.