Anti ccr6 pe cy7

Flow cytometry was performed as described using the following antibodies. The anti-CD3-FITC, anti-CD161-APC, and anti-TCR Vα7.2-Brilliant Violet 421(Biolegend, USA) were used to identify MAIT cells. Anti-CD4-PE/Cy7, anti-CD8-Perp/Cy5.5, anti-CCR6-PE/Cy7, anti-CXCR6-PE, anti-IL-17A-PE, anti-Granzyme B-PE/Cy7, and anti-Perforin-PE were obtained from Biolegend (USA). Anti-CD69-PE/Cy7, anti-PD-1-PE, anti-IFN-γ-PE/Cy7, anti-TNF-α-PE were obtained from BD (USA). Appropriate isotype control antibodies were used for each staining combination. We used 7-aminoactinomycin D(7-AAD) (BD Biosciences) staining to identify dead cells. Human MR1 tetramers loaded with a potent MAIT cell ligand-5-OP-RU or 6-FP were gifted from professor Li Bai. Apoptosis was allowed to progress and two channels were used to detect annexin V- FITC and 7-AAD (BD Biosciences) to determine the proportions of apoptotic cells. Data acquisition was achieved on BD FACS Verse system (BD Biosciences), and results were analyzed using FlowJo7.6 analysis software.

The lymphocytes from the spinal cord tissues were isolated by Percoll (GE Healthcare, Chicago, IL, USA) density-gradient centrifugation and analyzed by flow cytometry. Cells were stained with fluorochrome-conjugated monoclonal antibodies; mouse anti-CD45-Pacific blue (1:1000 diluted), anti-CD4-PE-Cy7 (1:1000 diluted), anti-CD8-PerCP-Cy5.5 (1:1000 diluted), anti-CD25-PE (1:1000 diluted), anti-CD69-FITC (1:1000 diluted), anti-CD44-APC-Cy7 (1:1000 diluted), anti-CXCR3-FITC (1:200 diluted), anti-CCR6-PE-Cy7 (1:200 diluted, BioLegend, San Diego, CA, USA), anti-IFNγ-FITC (1:100 diluted), anti-IL-17A-PE (1:200 diluted), and anti-FOXP3-APC (1:400 diluted, eBioscience, San Diego, CA, USA) and anti-T-bet-PE (3 µl per sample, BD, Franklin Lakes, NJ, USA). Intracellular cytokine staining was performed using an Intracellular Fixation and Permeabilization kit according to the manufacturer's instructions (eBioscience, San Diego, CA, USA). The samples were run on a BD Canto II cytometer (BD Biosciences, San Jose, CA, USA) and the results were analyzed by FlowJo software version 10.1 (BD, Franklin Lakes, NJ, USA).