Isolation and Culture of Antigen-Specific B Cells

PBMCs were isolated using Ficoll-Paque and stained with Live/dead Fixable Aqua dead cell stain kit 405 nm (life technologies), CD3-PB (UCHT1, BD), CD14-PB (M5E2, BD), CD19-APC-Cy7 (SJ25C1, BD), CD20-AF700 (2H7, Biolegend) and CD27-PE-Cy7(M-T271, BD). Antigen-specific staining was performed using CCP2-BV605, CCP2-APC and CArgP2-PE, or HC55-APC and HC55-PE [25 (link)] or TT-APC and TT-PE. Antigen-specific single B cell sorts were performed on a BD FACSAria III 4 L cell sorter at the Flow cytometry Core Facility (FCF) of the Leiden University Medical Center (LUMC) in Leiden, Netherlands. The cells were cultured on irradiated CD40L L cells in complex RMPI medium for 10–12 days [26 (link)]. RNA isolation, cDNA production, ARTISAN PCR and sequencing was performed as previously described [27 (link), 28 (link)].