Mil 21

3T3 cells expressing mouse CD40L and human BAFF (40LB cells Nojima et al., 2011 (link)) were maintained in Roswell Park Memorial Institute 1640 (RPMI-1640) media supplemented with 10% FBS and 1 x GlutaMAX (Gibco: 35050061). Irradiated (~120Gγ) 40LB cells were seeded at 80 × 10³ cells per well in a 12-well plate in supplemented RPMI-1640 media. The next day, naive B cells were isolated from the spleens of mice using the B cell isolation kit (Miltenyi Biotec: 130-090-862) and 30 × 10³ cultured on the irradiated 40LB cells with IL-4 in supplemented RPMI-1640 media (final: 10% FBS, 1 x GlutaMAX, 50 µM 2-mercaptoethanol Thermo Scientific: 31350–010, 100 units/ml penicillin, and 100 μg/ml streptomycin Thermo Scientific: 15140, 10 ng/ml mIL-4 PeproTech 214–14). On day 4 B cells were reseeded on freshly irradiated 40LB cells with IL-21 in supplemented RPMI-1640 media (final: 10% FBS, 1 x GlutaMAX, 50 µM 2-mercaptoethanol, 100 units/ml penicillin, and 100 μg/ml streptomycin, 10 ng/ml mIL-21 PeproTech 210–21). On day 8, B cells were harvested and analysed. 40LB cells were a kind gift from Prof D. Kitamura, they were negative for mycoplasma contamination, and authenticated for CD40L expression by flow cytometry.