B Cell Stimulation and Antibody Production

Cells were incubated in 50 μL of FACS buffer containing a cocktail of antibodies for 30 min on ice prior to washing and analysis on a FACS Aria (BD). Antibodies included anti-IgM FITC (G20-127, BD), anti-CD19 BUV395 (SJ25C1, BD), anti-CD3 BV711 (UCHT1, BD), anti-CD14 BV711 (M0P-9, BD), anti-CD16 BV711 (3G8, BD), anti-CD20 BUV737 (2H7, BD), anti-IgD BV605 (IA6-2, BD), and a fixable viability dye (Tonbo Biosciences, cat#13-0870-T500). B cells were individually sorted into flatbottom 96-well plates containing feeder cells that had been seeded at a density of 28,600 cells/well one day prior in 100 µL of IMDM media (Gibco, cat#31980030) containing 10% fetal calf serum, 100 U/mL penicillin plus 100 µg/mL streptomycin, and 2.5 µg/mL amphotericin. These 3T3 feeder cells produce CD40L, IL2, and IL21 to stimulate antibody secretion into culture supernatants [19 (link)]. B cells sorted onto feeder cells were cultured at 37 °C for 13 days.