2.3. Isolation and culture of human PBMC, B cells, and DC

Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood samples by gradient centrifugation according to the protocol provided by GE‐Healthcare (Ficoll‐Paque PLUS). B cells (CD19⁺) and monocytes (CD14⁺, CD16^‐) were isolated from PBMC by negative selection using the EasyEights™ EasySep™ Magnet (18103), the EasySep™ Human B Cell Isolation Kit (17954), and the EasySep™ Human Monocyte Isolation Kit (19359) all provided by StemCell™ Technologies.

B cells were maintained for up to 2 days in Roswell Park Memorial Institute medium (RPMI) 1640 medium (R8758, Roswell Park Memorial Institute medium; Sigma‐Aldrich), supplemented with 10% fetal bovine serum (FBS, 10270–106), 1% antibiotic‐antimycotic solution (AA, 15240062), and 1% l‐glutamine solution (l‐Gln, 11500626) all provided by Gibco, Thermo Fisher Scientific.

Monocytes were seeded and differentiated to dendritic cells (DC) in complete RPMI 1640 medium containing 10% FBS, 1% AA, 1% l‐Gln and supplemented with 800 IU/ml recombinant GM‐CSF (Granulocyte macrophage colony‐stimulating factor) and 500 IU/ml recombinant IL‐4 (R&D Systems) for 6 days at 37°C and 5% CO₂.