Isolation of CD19+ B cells from PBMCs

Isolation methods were previously described by us or other colleagues [22 (link),25 (link),47 (link)]. Briefly, buffy coats of healthy individuals obtained from the Swiss Red Cross Blood Donation Center Basel were centrifuged on Ficoll–Paque Plus (GE Healthcare Biosciences, Chicago, IL, USA) to separate PBMCs by a density gradient. To remove adherent cells, the collected PBMCs were incubated for 30 min at 37 °C in RPMI 1640 medium (#11875093, Gibco, Life Technologies, Carlsbad, CA, USA) (supplemented with 1% penicillin/streptomycin and 10% FBS depleted of extracellular vesicles) before the magnetic separation. Using anti-CD19-antibody-coated magnetic beads (#130-050-301, Miltenyi Biotec, Bergisch Gladbach, Germany), CD19+ B cells were isolated from the nonadherent PBMCs by positive selection according to the manufacturer’s instructions. The purity of the B-cell isolation was determined by flow cytometry with anti-CD20 (anti-CD20 FITC (#302304) or anti-CD20-PE (#302305), BioLegend, San Diego, CA, USA). Purity values were generally > 90%.