Anti cxcr6 pe

RPMI-1640 medium containing 10% fetal calf serum was routinely used for primary cell cultures. KnockOut Serum Replacement (Thermo Fisher, Waltham, MA, USA) was used for all migration assays involving S1P. Anti-CD4-PE-Cy7 (GK1.5), anti-CD4-FITC (RM4–5), anti-CD8-PE (53-6.7), anti-CD8-PECF594 (53-6.7), anti-CD69 PerCP-Cy5.5 (H1.2F3), anti-CD44-PE (IM7), anti-CD62L-APC (MEL-14), and anti-p-Stat3-Percp cy5 were purchased from BD PharMingen (San Diego, CA, USA). Anti-CCR2-PE-Cy7 (SA203G11), anti-CD45.2-FITC (104), anti-CD45.1-APC (A20), anti-Qa-2-biotin (695H1-9-9), anti-Qa-2-Alexa Fluor 647 (695H1-9-9), anti-Ki67-PE-Cy7, anti-CXCR3-APC, and anti-CXCR6-PE were purchased from BioLegend (San Diego, CA, USA). Anti-CCR2-APC (Catalog # FAB5538A) and anti-S1P1-PE (Catalog # FAB7089P) were purchased from R&D Systems (Minneapolis, MN, USA). Unlabeled antibodies against Akt, p-Akt (Ser473), STAT3, p-STAT3 (Tyr705), FoxO1 (C29H4), and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Recombinant mouse CCL2, CXCL9, and CXCL16 were purchased from R&D Systems (Minneapolis, MN, USA). Stat3 inhibitor Stattic was purchased from Selleck.

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.