Facs aria fusion

Cells were stained at 4 °C in the presence of Fc Block (2.4G2; BioXcell) in flow cytometry buffer (0.5% BSA in PBS). The following antibodies were purchased from Becton Dickinson (BD): PE-conjugated anti-CTLA-4 (UC10-4F10-11); PE-Cy7–conjugated anti-CD25 (PC81); biotin-conjugated anti-CD8b (53-5.8), from BioLegend: Pacific blue-conjugated anti-CD4 (RM4-5); PerCP/Cy5.5–conjugated anti-Thy1.1 (OX-7); Brilliant Violet421–conjugated anti-human CD4 (OKT4); APC-conjugated anti-human CD4 (RPA-T4), biotin-conjugated anti CD45R/B220 (RA3-6B2), from eBioscience: APC-conjugated IL-10 (JESS-16E3); efluor660-conjugated anti-GATA-3 (TWAJ); PE-conjugated anti-IRF4 (3E4), biotin-conjugated anti-CD49b (DX5), from Tonbo Biosciences: FITC conjugated anti-CD4 (GK1.5), from Invitrogen, R-PE-conjugated anti-human CD4 (S3.5). For IL-10 and CTLA-4 staining, cells were fixed in 2% paraformaldehyde for 15 min at RT and permeabilized with 0.5% Saponin before staining. For BATF, GATA-3 and IRF4, cells were fixed and permeabilized with Foxp3 Staining Buffer Set (eBioscience) following manufacturers’ instructions. Cells were analyzed on a FACSCanto II or FACS Aria Fusion and data were analyzed with FlowJo software (TreeStar).

Contaminating red blood cells were lysed using ACK Lysing Buffer. The remaining cells were washed in FACS buffer (1X PBS with 2% heat-inactivated fetal bovine serum (HI-FBS)). After staining, all cells were washed once in FACS buffer and resuspended in the same buffer containing 2 mg/mL propidium iodide (PI) to allow dead cells to be excluded. The samples and the data were acquired in a FACSCantoII Flow Cytometer, sorted in a FACSAria Fusion and analyzed using FlowJo software (TreeStar). Preincubation of cells with CD16/CD32 (2.4G2) Fc-block solution (BD Biosciences) was used to avoid nonspecific antibody binding. BM proB cells were purified in a FACSAria Fusion (BSII, Becton-Dickinson) as B220⁺ CD19⁺ c-Kit⁺ CD25⁻ PI⁻. The different antibodies used and the surface markers defining the different populations are indicated in Supplementary Experimental Procedures.