Antibiotin magnetic bead isolation

WT, Mb1-cre⁺ MyD88^fl/fl (B-MyD88⁻), WT-c-Myc-GFP, and Mb1-cre⁺ MyD88^fl/flMyc-GFP^+/+(B-MYD88⁻ c-Myc-GFP)naive B cells were isolated using Dynabead negative CD43 selection kit (Thermo Fisher Scientific) and stimulated in RPMI 1640 supplemented with 10% FCS, HEPES, 2-ME, and L-glutamine with anti–IgM-CpG and anti–IgM-Non for 24 h, then stained for viability before being analyzed by flow cytometry or fixed before staining for phospho-flow. Eμ-BCL2 Myc-GFP B cells were isolated by anti-CD11c, anti-CD43, and anti-IgD (GC) or anti-GL7 (naive) biotin Abs, followed by an antibiotin magnetic bead isolation (Miltenyi), according to a previously published protocol (21 (link)). Purity was confirmed at least 95% B220⁺ for naive B cells and 90% Fas⁺, CD38² for GC B cells. Cells were stimulated with 10–20 μg of anti-CD40 (FGK4.51; Miltenyi), 10–20 μg of anti-IgM (goat polyclonal μ chain; Jackson ImmunoResearch), 75 nM CpG 1826 oligo (IDT), or combinations of these reagents for 4 h in complete RPMI 1640 medium and processed for flow cytometry.