Anti igm f ab 2 abs

Bone marrow cells were extracted from femurs and pelvises of euthanized mice, and single-cell suspensions were incubated for 3 min in ACK lysis buffer (0.15 M NH₄Cl, 0.01 M KHCO₃, and 0.1 mM EDTA [pH 7.2–7.4]) to remove erythrocytes. B cells were enriched by positive selection using anti-B220 mAbs coupled to magnetic beads (Miltenyi Biotec) and an AutoMACS (Miltenyi Biotec) according to the manufacturer’s instructions. B cell purity was consistently >95% based on B220 staining. Enriched B cells (or total bone marrow cells for experiments with ERK, AKT, and mTORC1 inhibitors) were cultured at 2 × 10⁶ cells/ml at 37°C, 5% CO₂, in complete IMDM (5% FBS, 1% GlutaMAX, 1% penicillin-streptomycin, 1% nonessential amino acids, 0.1 M 2-ME) for times indicated in the figure legends. The following inhibitors were added to the bone marrow B cell cultures: actinomycin D (0.1 μM; Sigma-Aldrich), afuresertib (5 μM; Sigma-Aldrich), bafilomycin (12 nM; Sigma-Aldrich), bortezomib (0.5 μM; Selleck Chemicals), cycloheximide (2 μM; Sigma-Aldrich), FR180204 (30 μM; Santa Cruz Biotechnology), and rapamycin (10 μM; EMD Millipore). The following Abs were added for B cell stimulation: anti-3–83Ig idiotypic Ab S23 (49 (link)) (produced in-house) at ≤10 μg/ml (as indicated in figures) and anti-IgM F(ab’)₂ Abs (SouthernBiotech) at 5 μg/ml. DMSO and/or PBS diluents were added in control cultures.