Mouse primary B cell isolation and culture

Splenic B cells were isolated by negative selection as previously reported². Briefly, splenic B cells were isolated by anti-CD43 Ab-mediated negative selection, using a magnetic bead kit (Miltenyi Biotec, Auburn, CA) or mouse B cell isolation kit (STEMCELL, Vancouver, Canada), according to manufacturers’ protocols. Splenic T cells were isolated using a T cell isolation kit (STEMCELL), following the manufacturer’s protocol. Cells, including cell lines, were maintained in RPMI 1640 medium (Life Technologies, Grand Island, NY) containing 10 μM 2-β-mercaptoethanol (Sigma), 10% heat-inactivated FCS (Atlanta Biologicals, Atlanta, GA, USA), 2 mM l-Glutamine (Life Technologies), and 100 U/ml of penicillin-streptomycin antibiotics (Life Technologies). In glucose deprivation assays, cells were washed in sterile phosphate buffered saline (PBS, Life Technologies) to remove residual glucose-containing medium and incubated in glucose free RPMI 1640 medium supplemented by glutamine and 1 mM sodium pyruvate (Life Technologies) and 0.1% bovine serum albumin (BSA).