Efferocytosis Assay for B Cells

CD23⁺ B cells were purified from single cell suspensions of splenocytes with biotinylated anti-CD23 (BD Bioscience; #553137) and streptavidin microbeads (Miltenyi Biotec; #130-048-101) according to manufacturer’s instructions. Cells were cultured for 2–3 d in RPMI 1640 medium (Corning) supplemented with non-essential amino acids (Corning), 2 mM l-Glutamine (Corning), 25 mM HEPES (pH 7.2–7.6), and 50 μM β-Mercaptoethanol and stimulated with 5 μg/mL F(ab’)₂ anti-mouse IgM (Jackson ImmunoResearch), 5 μg/mL purified anti-mouse CD40 (BioXcell), and 50 ng/mL IL-21 (Peprotech). For thymocyte engulfment assays^{68 (link)} thymocytes were harvested and isolated from 4–6 week-old WT mice and treated with 50 μM Dexamethasone for 4 h to induce apoptosis. Apoptotic thymocytes were then stained with 1 μM CypHer5E (GE Healthcare) for 45 min at 37 ^oC in serum-free Hank’s Balanced Sodium Solution (HBSS). Stained thymocytes were co-cultured with splenocytes at a 1:10 splenocyte to thymocyte ratio. Apoptotic thymocytes were removed by washing with cold PBS and splenocytes were assessed for efferocytosis by flow cytometry. MHC-II expression was compared on efferocytic (CypHer5E⁺) and non-efferocytic (CypHer5E⁻) B cells.