Anti cd40 pe

The phenotype of pDC and CD1c⁺ mDC populations was determined by flow cytometry. DC purity was assessed by double staining CD11c⁺/CD1c⁺ for CD1c⁺mDCs (above 95%) and BDCA2/CD123 for pDCs (above 95%; all Miltenyi Biotec) [56 (link)]. The following primary monoclonal antibodies (mAbs) were used to determine the maturation state of the DCs: anti–CD80-APC, anti–PD-L1-APC (all BD Bioscience Pharmingen, San Jose, CA); and anti–CD40-PE (Beckman Coulter, Marseille, France). Measurements were performed on FACSCalibur and FACSVerse flowcytometers (BD).

CD1c⁺ DCs and pDCs were incubated overnight at 37°C with different stimuli in a 96-well round-bottom plate, either separate or together at a 1:1 ratio, with each well containing equal cell numbers (50 × 10³ cells in 100 μL). After overnight culture, supernatants were taken and cells were stained with the following primary monoclonal antibodies: anti-human leukocyte antigen (HLA)-ABC-APC (BD Biosciences, 555555), anti-HLA-DR/DP/DQ-FITC (BD Biosciences, 555558), anti-CD80-PE (BD Biosciences, 557227), anti-CD83-FITC (BD Biosciences, 556910), anti-CD83-APC (BD Biosciences, 551073), anti-CD86-PE (BD Biosciences, 555658), anti-PDL-1-PE (BD Biosciences, 557924), anti-CD40-PE (Beckman Coulter, PN IM1936U), anti-CCR7-PE (Miltenyi Biotec, 130-093-621). Anti-CD11c-PE (BD Biosciences, 333149) or anti-CD123-APC (Miltenyi Biotec, 130-090-901) primary monoclonal antibodies were used to distinguish between DC subsets in co-cultures. Samples were measured on a FACSCalibur or FACSVerse (BD Biosciences) and analyzed by FlowJo software (TreeStar, Inc.). The results are depicted as geometric mean fluorescence intensity (MFI) normalized to the negative control. Supernatants were analyzed for IL-12p70 (Thermo Fisher Scientific, M122), TNF-α (eBioscience, 88-7346-88) and IFN-α (Bender Medsystems, BMS216MST) by sandwich ELISA.

Purity of pDCs and mDCs after isolation and the phenotype of the pDC populations were determined by flow cytometry. The following primary monoclonal antibodies (mAbs) and the appropriate isotype controls were used: anti-BDCA1-FITC, BDCA2-PE, BDCA4-PE and CD123-APC (all Miltenyi Biotec); mIgG1-PE, mIgG1-APC, anti-CD11c-FITC or -APC, anti-HLA-ABC-PE (W6/32), anti-CD80-PE, or -APC, or -PeCy7, anti-CD86-PE, or -APC (all BD Bioscience Pharmingen, San Diego, CA, USA) anti-PD-L1-APC, anti-PD-L2-PE; anti-CD40-PE, anti-CD83-PE (Beckman Coulter, Mijdrecht, the Netherlands); anti-MHC-II-APC (eBioscience).
The phenotype of the DC populations after treatment with vemurafenib, dabrafenib, trametinib or a combination was determined by staining with the following antibodies and appropriate isotype controls: anti-CD80-PE-Cy7, anti-CD86 APC, anti-PD-L1-PE, anti-HLA-ABC-V450, anti HLA-DR-BV510, mIgG1-PE-Cy7, mIgG1-PE (all BD biosciences) and mIgG1-APC (eBioscience).