Cd21 fitc

Antihuman: p-BTK (pY223) PE, CD19 PE-Cy5, IgM PE, CD45 AmCyan and CD45 FITC (Becton Dickinson, USA), CD39 PE-Cy7, CD73 FITC, CD73 eFluor450, CD21 FITC and CD23 APC (eBioscience, USA). Anti-mouse: B220 FITC, CD4 PerCP, CD45 APC-Cy7 (BD-Pharmingen, USA), IgM PE (Southern Biotech, USA) and CD3e eFlour450, CD39 PE-Cy7, IgD FITC, IgM APC (eBioscience, USA), IgD APC-Cy7, CD73 PacificBlue, CD19 PE-Cy7 and CD8a PE (BioLegend, USA). All human mAbs were titrated using normal PBMC to establish optimal dilution.

Cell suspensions of different organs were lysed with ammonium chloride buffer (0.150 mM NH₄Cl, 0.1 mM EDTA, 0.150 mM KHCO₃) to eliminate erythrocytes followed by staining with CD19-FITC/APC (eBio1D3, 1:100), CD38-PE (clone 90, 1:100), IgM-PE/PECy7 (II/41, 1:200), IgD-eFlour450 (11-26c, 1:30), CD5-APC/Pacific Blue (53-7.3, 1:80), B220-eFlour605 (RA3-6B2, 1:40), CD3-FITC (145-2C11, 1:100), ZAP70-FITC (1E7.2, 1:20), CD21-FITC (eBio8D9, 1:100), CD23-PECy7 (B3B4, 1:160), CD93-PE (AA4.1, 1:80), LIN-Cocktail (CD11b-Biotin (M1/70, 1:130), CD3-Biotin (145-2C11, 1:130), Ter119-Biotin (Ter119, 1:130) and Gr1-Biotin (RB6-8C5, 1:130), Streptavidin-PerCP (1:160), Annexin V-Pacific Blue (1:25) and 7-AAD (1:100) (all from eBioscience). Cells were analysed with the Canto II cytometer (BD Bioscience). Data was obtained with the BD FACSDiva^TM (BD) and analysed with the FlowJo 8.5.3 software. The sequential gating strategy for all FACS panels is provided in Supplementary Fig. 11.