Cd20 apc h7 clone 2h7

Whole blood was collected on EDTA, then transferred into BD vacutainer CPT tubes (after removal of anticoagulant solution from CPT tubes) and spun at 2500g for 20 min. The PBMCs were collected and one-tenth were used to isolate RNA. The remaining cells were surface stained on ice using three different panels, including Panel A: CD150 BV-421 (clone A12, BD), CD8 AF 647 (clone RPA-T8), CD20 APC-H7 (clone 2H7, BD), CD3 V500 (clone SP34–2, BD), CD14 Pe-cy7 (clone M5E2, BD); Panel B: CD150 BV421, CD3 V500, CCR7 Pe-Cy7 (clone G043H7, Biolegend), CD8 AF647, CD45RA APC-H7 (clone 5H9, BD); and Panel C: CD150 BV421, IgD BV510 (clone IA6–2, BD), CD38 Pe-Cy7 (clone HB7, BD), CD27 AF647 (clone O323, Biolegend), CD20 APC-H7. Cells were acquired on a MACSQuant®10 flow cytometer (Miltenyi).

Whole blood was collected on EDTA, then transferred into BD vacutainer CPT tubes (after removal of anticoagulant solution from CPT tubes) and spun at 2500 g for 20 min. The PBMCs were collected and one-tenth were used to isolate RNA. The remaining cells were surface stained on ice using three different panels, including Panel A: CD150 BV-421 (clone A12, BD), CD8 AF 647 (clone RPA-T8), CD20 APC-H7 (clone 2H7, BD), CD3 V500 (clone SP34-2, BD), CD14 Pe-cy7 (clone M5E2, BD); Panel B: CD150 BV421, CD3 V500, CCR7 Pe-Cy7 (clone G043H7, Biolegend), CD8 AF647, CD45RA APC-H7 (clone 5H9, BD); and Panel C: CD150 BV421, IgD BV510 (clone IA6-2, BD), CD38 Pe-Cy7 (clone HB7, BD), CD27 AF647 (clone O323, Biolegend), CD20 APC-H7, using 4 µl of each Ab per sample. Cells were acquired on a MACSQuant®10 flow cytometer (Miltenyi) and analyzed by FlowJo10.8 (Becton Dickinson) and Kaluza 2.1 (Beckman Coulter). Gating strategy is presented in supplementary fig. S8.

To determine the phenotype of B‐cells post‐stimulation, cells were stained with LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Thermo Fisher, Waltham, MA, USA) and then with anti‐human IgM BV605 (clone MHM88; Biolegend), IgG BV786 (clone G18‐145; BD), CD27 APC (clone 323; Biolegend), CD20 APC‐H7 (clone 2H7; BD), CD38 PE (clone HIT2; BD), CD19 PE‐CF594 (clone HIB19; BD), CD71 PE‐Cy7 (clone CY1G4; Biolegend), IgD BUV395 (clone IA6‐2; BD) and CD21 BUV737 (clone B‐Iy4; BD). The dump channel mix consisted of BV510‐labelled anti‐human CD3 (clone OKT3), CD10 (clone HI10a), CD14 (clone M5E2) and CD16 (clone 3G8), all from Biolegend. Samples were stained according to standard techniques and fixed briefly with 1% formaldehyde before acquiring data on an LSRFortessa flow cytometer (BD Biosciences). Data were analysed using FlowJo v10.5.3 (Tree Star, Inc., Ashland, OR, USA).