Ter119 bio

Splenic naive B cells were purified with the MagniSort Streptavidin Negative Selection Beads (eBioscience) following the manufacturer’s instructions, using TCRb-Bio (BioLegend, 109204), CD11b-Bio (Biolegend, 101204) and Ter119-Bio (Biolegend, 116204). iGC B cell culture was performed following (Nojima et al., 2011 (link)). Briefly, B cells were plated (5 × 10⁶ cells per 10 cm dish) on mitomycin C (Sigma, M0305)-treated 40LB feeder cells (3 × 10⁶ cells per 10 cm dish) in 40 mL B cell medium: DMEM (Sigma, WHMISDZB)) supplemented with 10% (v/v) FBS (GIBCO, 10270-106), 2 mM L-glutamine (Sigma, G7513), 10 mM HEPES (LONZA, BE17-737E), 1 mM sodium pyruvate (GIBCO, 13360-039), 1 x non-essential amino acids (GIBCO, 11140-035), 100 U/ml penicillin, 100 μg/ml streptomycin (Sigma, 0781), 50 μM β-mercaptoethanol (Sigma, M3148) and 10 ng/ml rIL-4 (Peprotech, 214-14); 30 mL of medium were changed on day 3. On day 4, iGC B cells were harvested, analyzed and 1,5 × 10⁶ cells replated per 10 cm dish onto a new Mitomycin C-treated feeder layer in B cell medium supplemented with either 10 ng/ml rIL-4 or 10 ng/ml rIL-21 (Peprotech, 210-21); 30 mL of medium were changed on day 7 and the final analysis was performed on day 8. The analysis included cell count with the trypan blue exclusion method using a hemocytometer (Neubauer) and flow cytometric analysis, as described above.