Isolating and Culturing Lymphoid Cells

For splenocyte and lymph node cell preparation, organs were mashed through a 70 μM cell strainer (BD Biosciences), as previously described (Rosser et al., 2014 (link)), and erythrocytes from spleens were lysed using Red Cell Lysis Buffer (Sigma-Aldrich). B cells were negatively purified by magnetic separation, according to manufacturer’s instructions (Miltenyi Biotec). Cells were cultured with either RPMI 1640 (Sigma-Aldrich) containing L-glutamine and NAHCO₃ or Iscove’s Modified Dulbecco’s Medium (IMDM; Pan Biotech), enriched in AhR agonists (Veldhoen et al., 2009 (link)), supplemented with L-Glutamine and 25mM HEPES. Media were supplemented with 10% fetal calf serum (LabTech), 1% Penicillin/streptomycin (100U/ml Penicillin+100 μg/ml streptomycin; Sigma-Aldrich) and 50 μM 2-Mercaptoethanol (ThermoFisher Scientific). Cells were cultured at 37°C with 5% CO₂.
Total lymphocytes, B cells and B cell subsets were cultured for 48h with CpGb ODN1826 (1 μM; Invivogen), LPS (1 μg/ml; Sigma-Aldrich) ± anti-mouse IgM (10 μg/ml; Jackson ImmunoResearch). or anti-CD40 (10 μg/ml; BioXcell). In addition, AhR agonist FICZ (100nM; Enzo LifeSciences) was added to culture. For 48h culture, anti-IgM ± FICZ were added 24h into culture.