Anti cd40 antibody

MAD2L2 WT, MAD2L2 R124A, and MAD2L2 3xMut(K44A/R124A/A135D) vectors were introduced by retroviral infection and CH12 cells were selected with blasticidin (10 μg ml⁻¹). The aforementioned TRIP13-shRNA and control vectors were introduced by lentiviral infections and CH12 cells were selected with puromycin (3 μg ml⁻¹). CH12 cells were subsequently plated at 50,000 cells/ml in complete RPMI supplemented with anti-CD40 antibody (1 μg ml⁻¹, Miltenyi), IL-4 (20 ng ml⁻¹, Miltenyi) and TGF-β (1 ng ml⁻¹, R&D Biotech) to induce IgM to IgA switching. After 3–4 days, cells were assayed for class-switching by flow cytometry using an IgA-PE antibody (eBiosciences) and a Fortessa analyser (BD Biosciences). Viable cells were counted using a Casy cell counter (Roche). CSR and proliferation assays were done in at least three independent experiments.

B cells were isolated using B cell isolation kit (Milteny Biotec, Germany) and cultured in RPMI 1640 medium (Gibco) with 10% FBS, 1% penicillin/streptomycin and 50 µM β-mercaptoethanol. 3 × 10⁶ cells were cultured in 3 ml medium supplemented with 10 μg/ml anti-CD40 antibody (Milteny Biotec, Germany) and 30 ng/ml IL-4 (PeproTech, Germany) for 7 days. On day 4, cells were collected and analyzed for the presence of CD138⁺ B220^low ASCs by flow cytometry. On day 7, supernatants were harvested. To determine immunoglobulin levlels, 96-well plates were coated with 2 μg/ml of goat anti-mouse IgM and IgG1, overnight at 4°C. Subsequently, plates were blocked with 10% FCS in PBS for 2 h at RT. Supernatants were added in duplicates. Bound antibodies were detected using alkaline phosphatase-conjugated goat anti-mouse IgM or IgG1 (SouthernBiotech, United States) diluted 1:1000. 4-nitrophenil phosphate-disodium salt (Serva, Germany) was used as substrate. IgM and IgG1 serum titers were determined by using respective isotype-specific Ig standards (SouthernBiotech, United States).

CH12 cells were plated at 50,000 cells per ml in complete RPMI supplemented with anti-CD40 antibody (1 μg/ml, Miltenyi), IL-4 (20 ng/ml, Miltenyi), and TGF-β (1 ng/ml, R&D Biotech) to induce IgM to IgA switching. After 3 days, cells were assayed for class switching by flow cytometry using IgA-PE (eBiosciences 12-4204-82, clone mA-6E1, 1:200) and IgM-PE-Cy7 (BD Biosciences 552867, clone R6–60.2, 1:200 dilution) antibodies, and a Fortessa analyser (BD Biosciences). Data were analyzed by FlowJo v10.4.2 software. The gating strategy is provided in Supplementary Fig. 8.