B Cell Switching Protocols for CSR

Spontaneous switching human IgM⁺ IgD⁺ 4B6 B cells, inducible switching human IgM⁺ IgD⁺ 2E2 B cells and inducible switching mouse IgM⁺ CH12F3 B cells were as we described12 (link). Human IgD⁻ CD38⁺ CD19⁺ germinal center B cells and IgD⁺ CD38⁻ CD19⁺ naïve B cells from tonsil and mouse B220⁺ primary B cells were prepared as we described12 (link), 68 (link). To analyze spontaneous CSR in human 4B6 B cells, IgG⁻ IgA⁻ cells were cultured at 5 × 10⁴ cell ml⁻¹ in RPMI-1640 medium (Invitrogen Corp.) supplemented with 10% fetal calf serum (Hyclone) and 1% penicillin-streptomycin (FCS-RPMI). To induce CSR to IgG1, human 2E2 B cells were cultured at 5 × 10⁴ cell ml⁻¹ and stimulated with agonistic mAb to hCD40 (IgG1 mAb G28-5, ATCC) plus recombinant hIL-4 (Genzyme Co.) for 4 d. Mouse B cells were labeled with CFSE (Invitrogen) and induced to undergo CSR by mCD154 or LPS (E. coli serotype 055:B5; Sigma-Aldrich Co.) plus appropriate cytokines, as we reported12 (link). All protocols involving human tissues and/or cells were in accordance to the rules and regulations of the IRB of UC Irvine.