Gr1 biotin

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.

For the flow cytometry analyses, cells were stained with monoclonal antibodies against the following anti-mouse molecules: CD3-biotin, Ter119-biotin, GR-1-biotin, B220-biotin, CD11b-biotin, NK1.1-biotin, CD3-biotin, CD4-biotin, CD8-biotin, cKit-BV650, Sca1-PE, CD135-PerCP, CD34-APC, CD25-PE, CD44-APC, CD3-APC, CD8-PerCP, CD-BV650, CD45.1/Ly5.1-PE-Cy7, CD45.2/Ly5.2-APC-Cy7 (all from eBiosciences, CA, USA). For secondary detection of biotinylated antibodies, streptavidin conjugated with FITC or PE-Cy7 was used (eBiosciences, San Diego, CA, USA). All flow cytometry measurements (Canto II BD biosciences or LSRII BS Biosciences) were calibrated first with BD™ CompBead Plus, κ/Negative Control (BSA) Compensation Plus (7.5 µm). Recommended flow cytometry settings are specified in [17 ]. Flow cytometry analyses were performed using FlowJo software (Treestar, Ashland, OR, USA).