Mature, resting B cells were obtained from mouse spleens by forcing tissue through a 70-µm mesh into PBS containing 2% heat-inactivated fetal bovine serum (FBS). After ammonium-chloride-potassium buffer lysis for 3 min, untouched B cells were enriched using anti-CD43 magnetic beads according to the manufacturer’s protocol (Miltenyi Biotec) obtaining >95% purity. 3.2 × 107 cells/10 cm dish (Gibco) were cultured at 37°C 5% CO2 in 10 ml mouse B cell medium consisting of RPMI-1640, supplemented with 10% heat-inactivated FBS, 10 mM Hepes, antibiotic-antimycotic (1×), 1 mM sodium pyruvate, 2 mM L-glutamine, and 53 µM 2-mercaptoethanol (all from Gibco) and activated with 2 µg/ml anti-mouse RP105 clone RP/14 (produced in house or 562191; BD Pharmingen).
NB-21 feeder cells (Kuraoka et al., 2016 (link)) were maintained in DMEM supplemented with 10% heat-inactivated FBS and antibiotic-antimycotic (1×). For co-culture, feeder cells were irradiated with 80 Gy and seeded simultaneously with B cells, 24 h after transfection, into B cell culture medium supplemented with 1 ng/ml recombinant mouse IL-4 (214–14; PeproTech) and 2 µg/ml anti-mouse RP105 clone RP/14.
NB-21 feeder cells (Kuraoka et al., 2016 (link)) were maintained in DMEM supplemented with 10% heat-inactivated FBS and antibiotic-antimycotic (1×). For co-culture, feeder cells were irradiated with 80 Gy and seeded simultaneously with B cells, 24 h after transfection, into B cell culture medium supplemented with 1 ng/ml recombinant mouse IL-4 (214–14; PeproTech) and 2 µg/ml anti-mouse RP105 clone RP/14.