Il 12 p40 pe

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.

Cell surface antigens were stained first. The dendritic cell antibody panel quantified the following: CD11c-APC-eFluor780, CD80-PE-Cy7, CD86-FITC, and CD40-eFluor450 (all mAbs are from eBioscience now Thermo Fisher, Waltham, MA). The CD4+ T_Reg cell panel included the following: CD3ε-PE, CD4-PE-Cy7, and CD25-PerCP-Cy5.5s (all mAbs are from eBioscience). For T_Reg intracellular staining, cells were fixed and permeabilized with the one step eBioscience Fix-Perm Foxp3 Buffer Staining Kit (eBioscience). The panel included Foxp3-eFluor450 and CTLA4-APC (mAbs are from eBioscience). Intracellular staining was also used to detect TGFβ-PE, IL12 p40-PE, IL10-APC, and IFNγ-APC production (mAbs are from eBioscience) after ex vivo nonspecific antigen stimulation with a Leukocyte Activation Cocktail with GolgiPlug (BD Biosciences) for flow cytometry. Mouse INFγ Single-Color ELISPOT to determine antigen-specific response of T cells was from Cellular Technology Limited (CTL), Cleveland, OH. For attempting to assess SIRT1 expression by flow cytometry, we used antibodies from Santa Cruz and Abcam.