Cd45 percp cy5.5 30 f11

Cells were washed twice with DPBS and stained with either fixable viability dye eFluor506 (eBioscience) or fixable viability stain 780 (BD) and anti-CD16/CD32 (2.4G2; BD) in DPBS for 10 min at 4°C. Cells were then washed with FACS buffer containing 2% fetal calf serum (Invitrogen), 2.5 mM EDTA (MP Biomedicals) in PBS. For cell surface staining, cell pellets were re-suspended in antibodies diluted in FACS buffer and stained for 30 min at 4°C. Intra-nuclear staining was performed using the Foxp3 staining kit (eBioscience). Intracellular cytokine staining was performed with the Cytofix/Cytoperm staining kit (BD). The following mouse-specific conjugated antibodies were used: CD45-PerCP-Cy5.5 (30-F11; BD), CD45-BV510 (30-F11; BD), TCRβ-BV786 (H57-597; BD), CD4-Pe-Cy7 (RM4-5; BD), Helios-AlexaFluor488 (22F6; BD), CD3-PB (145-2C11; Biolegend), CD4-BV785 (RM4-5; Biolegend), Helios-FITC (22F6; Biolegend), IL10-Pe-Cy7 (JES5-16E3; Biolegend) Foxp3-AlexaFluor700 (FJK-16s; eBioscience), and RORγt-PE (Q31-378; eBioscience). Cells were acquired on a FACS Fortessa (BD Biosciences) or a FACS Canto (BD) with FACSDIVA software (BD). Data analysis was performed using FlowJo software (Tree Star Inc.).