Cd4 bv785 rm4 5

Unspecific binding was blocked by incubating cells with Fc receptor-blocking monoclonal antibody (clone 2.4G2; BD Biosciences) for 10 min. Cells were then stained with the following antibodies specifically binding: CD3-PerCP/Cy5.5 (145-2C11; BioLegend), CD4-BV785 (RM4-5; BioLegend), CD8-BV421 (53-6.7; BioLegend), B220/CD45R-APC-Cy7 (RA3-6B2; BioLegend), CD138-BV605 (281-2; BioLegend), CD69-PE (H1.2 F3; BioLegend), and PD-1-PE-Cy7 (29 F.1A12; BioLegend). Dead cells were excluded by ZombieGreen™ (BioLegend) staining, and doublets by scatter analysis. Cells were measured on a LSRII flow cytometer (BD) and analyzed by FlowJo software. In most samples, a minimum of 1 × 10⁵ events were measured.

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.).