Flow Cytometric Analysis

For surface marker analysis, cell suspensions were adjusted to a density of 1 × 106 cells in 50 μl FACS buffer (2% FBS, 0.05% NaN₃ in PBS). Non-specific binding was blocked by incubation for 10 min at 4°C with anti-CD16-CD32 antibody (BioLegend, clone 93, 5 ng/μl) and stained with the appropriate antibodies for 15 min at 4°C. The following antibodies were used for extracellular staining: CD45 (clone 30F-11, 0.5 ng/μl), CD4 (clone RM4-5, 0.5 ng/μl), TCR-γδ (clone GL3, 4 ng/μl), CD3 (clone 145-2C11, 2 ng/μl), CD25 (clone 3C7, 0.2 ng/μl) from BioLegend. For intracellular staining, cells were first stained for surface markers as detailed above, fixed and permeabilized using Fixation and Permeabilization buffers from eBiosciences following the manufactures' instructions. Briefly, cells were fixed for 30 min at 4°C, washed with Permeabilization Buffer and incubated for 30 min with IL-17A (clone eBio 17B7, 1 ng/μl) or FoxP3 (clone FJK-16 s, 0.5 ng/μl) antibodies in Permeabilization Buffer at 4°C. Cells were washed with FACS buffer, resuspended in 200 μl of FACS buffer and phenotyped on MACSQuant 10 cytometer (Myltenyi Biotec). Analysis of each sample was performed with FlowJo software (version 6, Tree Star).