Tnfα percpcy5

PBMCs were cultured in RPMI-1640 medium (GIBCO) containing 10% fetal bovine serum and stimulated with anti-CD3/CD28 (2 and 5 μg/ml) antibodies or phorbol 12-myristate 13-acetate (PMA)/ionomycin (50 ng/ml and 1 μg/ml, respectively), plus Golgiplug (BD Pharmingen, San Diego, CA, USA) for 5 h. The cells were then surface stained with CD4-FITC, CD8-APC-H7, PD-1-PE and TIM-3-PE-Cy7, and intracellularly stained with IFN-γ-PE-CF594, TNF-α-PerCp-Cy5.5 or IL-2-PerCp-Cy5.5 (BD Pharmingen) antibodies. A violet amine reactive dye (Invitrogen, Grand Island, NY, USA) was used to assess cell viability.

Chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO). Anti-mouse or anti-human fluorochrome labeled antibodies (Abs): CD4-PB, CD62L-APC, CD44-PerCP-Cy5.5, CD127-PE, FoxP3-FITC, Tim3-PE, PD1-PECy7, IFN-γ-PECy7, TNFα-PerCPCy5.5, IL-17-PerCPCy5.5, CD25APC-Cy7, CD45RA-PE, CD45RO-APC, and Abs for ELISA were procured from BD Pharmingen (San Diego, CA) or otherwise mentioned. RPMI-1640 and FBS were purchased from GIBCO (Grand Island, NY) for cell culture. For culturing of cells, tissue culture grade plastic-wares were purchased from BD Biosciences (Bedford, MA). Ab against iNOS used in Western blot was procured from (Abcam, Cambridge, United Kingdom).

Brefeldin A solution (eBioscience, CA, USA) was added to monocytes and macrophages cultures (10 μg/ml) 15 hours before the cells were harvested. Cells were stained with appropriate anti-human monoclonal antibodies such as CD14-FITC (eBioscience), CD16-BD Horizon PE-CF594 (BD Biosciences, New Jersey, USA), CD86-PE (eBioscience), CD11b-PE/Cy7 (eBioscience), HLA-DR-APC/Cy7 (BioLegend, CA, USA), PD-L1-APC (eBioscience), CD80-PerCP3Flour^®710 (eBioscience), CD163-APC (eBioscience), CD206-APC/Cy7 (Biolegend), CD64-PE (Biolegend), CD209-PE/Cy7 (eBioscience), T-bet-PerCP/Cy5.5 (eBioscience), IL-12(p40)-PE (eBioscience), IL-10-Alexa Fluor 647 (eBioscience) and TNFα-PerCP-Cy5.5 (BD Pharmingen^TM) following manufacturer’s protocol. Data were acquired on FACSAria III flow cytometer (BD Biosciences) and analyzed with FlowJo Version 7.6 (Ashland, OR, USA) based on 20,000 cells.