Leaf purified anti cd40 antibody hm40 3

Naive splenic B cells were purified by positive selection using CD23 coated microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) followed by magnetic selection of labeled cells (B cell purity > 95%). All primary mouse B cells were cultured in RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 10% fetal bovine serum (FBS, Hyclone, Thermoscientific, Logan, Utah), 2 mM L-glutamine, penicillin (50 U/ml), streptomycin (50 U/ml), pyruvate (1 mM), MEM NEAA (100 uM), 2-ME (55 uM), HEPES (10 mM). For experiments in which B cells were stimulated in Ca²⁺ free media, 0.5 mM EGTA was added to complete RPMI media.-For in vitro stimulation assays, BCR was engaged using soluble 10 mg/mL anti-mouse F(ab′)2 antibody (Jackson ImmunoResearch, West Grove, PA) unless indicated otherwise. For CD40 engagement, LEAF purified anti-CD40 antibody (HM40–3, BioLegend, San Diego, CA) was used at a final concentration of 2 ug/mL. CpG (ODN1826: 5′-TCC ATG ACG TTC CTG ACG TT-3′) was synthesized and high-performance liquid chromatography purified by Integrated DNA Technologies (Coralville, IA). CpG was used at a final concentration of 1 µM. BLyS (R&D Systems, Minneapolis, MN) was used at final concentration of 100 ng/mL.