B cell development and class switch recombination

B cell development in bone marrow and spleen of sex-matched Fam72a^−/− and Fam72a^+/+ littermates (6–8 weeks old) was evaluated (Supplementary Fig. 2) as described^{33 (link)}. To induce ex vivo CSR to different immunoglobulin isotypes, splenic B cells were purified with a mouse B cell isolation kit (StemCell Technologies), and then stimulated with LPS (Sigma-Aldrich) in combination with various cytokines, as previously described^{33 (link)}. Cells were collected at day 4 after stimulation and were stained with antibodies against mouse IgG1 (PE, BD Pharmigen, cat. no. 550083; 1: 150 dilution), IgG2b (PE, SouthernBiotech, cat. no. 1090–09S; 1:150), IgG3 (FITC, BD Pharmigen, cat. no. 553403; 1:100), IgE (FITC, BD Pharmigen, cat. no. 553415; 1:100) or IgA (PE, SouthernBiotech, cat. no. 1040–09; 1:150) to assess CSR. For the cell cycle analysis, splenic B cells were stimulated with LPS for 2.5 days and analysed with the Click-iT Plus EdU Alexa Fluor 647 kit (ThermoFisher Scientific) as described^{33 (link)}. IgG1 CSR was induced in carboxyfluorescin diacetate succinimidyl ester (CFSE)-pulsed splenic B cells as we previously described^{33 (link)}. The apoptosis of LPS stimulated splenic B cells was assayed using an APC-conjugated Annexin V Apoptosis Detection Kit (eBioscience). The flow cytometric data were analysed with a FlowJo X10 software.