Cd4 pecy7 rm4 5

PB was collected from the retro-orbital plexus in heparinized capillary tubes (Fisherbrand, Pittsburgh, PA) and lysed in red blood cell lysis buffer (Sigma-Aldrich, St. Louis, MO). Cells were stained with the following antibodies: CD45.1-APC (A20) (Biolegend, San Diego, CA) or CD45.1-FITC (A20) (BD Biosciences, San Diego, CA), B220-PECy7 and CD8-PECy7 (53–6.7) (Tonbo Biosciences, San Diego, CA), CD45.2-V500 (104), B220- PerCPCy5.5 (RA3-6B2), Gr1-PerCPCy5.5 (RB6-8C5), Cd11b-PerCPCy5.5 (M1/70) and CD4-PECy7 (RM4-5) (BD Biosciences, San Diego, CA). All antibodies were used at 1:200 dilution. 4’,6-diamidino-2-phenylindole (DAPI) staining was used to gate live events. Analysis was performed on a LSR Fortessa and a BD FACSAria III SORP (Special Order Research Product, which contains the following LASERs: 405 nm, 445 nm, 488 nm, 562 nm, and 640 nm) (both BD Biosciences, San Diego, CA) and the data analyzed with FlowJo version 9.4.11 (Tree Star, Ashland, OR). To discern the four Confetti colors: the filter arrangements for each LASER were: CFP, 445 nm LASER—470/24 band-pass filter (BP); GFP, 488 nm LASER—515/20 BP and 505 long-pass filter (LP); YFP, 488 nm LASER—545/10 BP and 525 LP; RFP, 562 nm LASER—610/20 BP and 600 LP^{48 (link)}.

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.).