Cd19 allophycocyanin hib19

B cells were isolated (negative selection), and purity was > 92% (n = 4; two donors from Barcelona and two donors from Stockholm). A total of 2–9 × 10⁶ B cells was labeled for 5 min at room temperature with 1.25 µM CFSE (CellTrace CFSE Cell Proliferation Kit; Invitrogen). Labeled B cells were washed three times (350 × g, 7 min) and cultured in complete culture medium at a density of 1.5 × 10⁵ cells/200 µl (96-well round-bottom plates; BD). B cells were either left unstimulated or stimulated with IL-21 (100 ng/ml; Invitrogen), MegaCD40L (500 ng/ml; Enzo Life Sciences), or 5 or 25 µg exosomes/well. After 5 d of cultivation (37°C, 5% CO₂), cells were incubated with purified human Ig (Sigma-Aldrich) and subsequently stained with CD19-allophycocyanin (HIB19), CD20-PerCP-Cy5.5 (2H7) and CD38-PeCy (HB7; all from BD). DAPI (5.7 µM; Sigma-Aldrich) was used as live/dead marker. Cells were analyzed with flow cytometry and gated as follows: FSC-A-SSC-A, FSC-A-FSC-H, DAPI⁻ (alive), CD19⁺.

B cells were isolated from PBMCs of healthy blood donors (Blood Transfusion Center Solna or Banc de Sang i Teixits, Barcelona, Spain). B cells were isolated either through negative selection (B Cell Isolation Kit II; Miltenyi Biotec) or by positive selection using biotinylated anti-IgD Ab (Southern Biotech) and Anti-Biotin MicroBeads (Miltenyi Biotec). The purity and B cell composition of each donor were assessed by flow cytometry, staining for CD19-allophycocyanin (HIB19; BD), IgD-FITC (IA6-2; BD), CD38-PECy (HB7; BD), CD27-PE (M-T271; BD), and DAPI (5.7 µM; Sigma-Aldrich) using an LSR II or Fortessa (BD) and analyzed using FlowJo software (TreeStar).