Pacific blue conjugated anti cd4 rm4 5

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).