Cd43 depletion

Splenic naive B cells were purified by CD43 depletion (Miltenyi). GC B cells were purified as previously described (Luo et al., 2018 (link)). Naive and GC B cells were cultured in vitro in the presence of 10 µg/ml (anti-IgM + anti-IgG; Jackson ImmunoResearch) and/or 20 µg/ml anti-CD40 antibody (FGK45; Bio X Cell) with or without 25 ng/ml IL-4. For cell proliferation studies, naive B cells were stained with CellTrace Violet (Thermo Fisher) according to the manufacturer’s protocol. HEK293T cells were transiently transduced with a MIZ1-IRES-GFP expressing vector or GFP control (VectorBuilder) using Lipofectamine (Invitrogen). Cells were analyzed 48 h after transduction.

Splenic B cells were purified from mice with CD43⁺ depletion (Miltenyi Biotec). Cells were cultured in complete media (RMPI-1640 supplemented with 10% FBS, 1% penicillin-streptomycin, 1% sodium pyruvate, 1% Hepes, 1% GlutaMAX, and 0.1% β-ME) for 48 h at 37°C. B cells were stimulated with or without the following reagents; R848 (5 ng/ml); anti-mouse IgM F(ab')₂ fragment (1 μg/ml; Jackson ImmunoResearch, Inc.); anti-mouse CD40 (1 μg/ml; SouthernBiotech); IL-12 (20 ng/ml). Supernatant was collected and evaluated with an IL-6 ELISA (eBioscience).