Anti cd83 pe

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).

EDTA whole blood samples from 5 healthy subjects, 10 patients with mild and 10 patients with severe disease were stained with anti-HLA-DR PE and ECD, anti-CD3 ECD, anti-CD4 PE, anti-CD8 FITC, anti-CD19 PC7, anti-CD14 FITC, anti-CD16 PC5, anti-CD15 PE, anti-CD57 FITC, anti-CD56 PE, anti-CD11c PCP, anti-CD123 PE, anti-CD83 PE, anti-CD38 PE, anti-CD23 ECD and isotype controls (all from Beckman Coulter) for 20 minutes in the dark at +4°C. Samples were analyzed on the flow cytometer Cytomics FC500 (Beckman Coulter). Data were processed by FlowJo V.10.

Detection of the cell surface expression of monocyte-derived myeloid cells was performed by flow cytometry using anti-CD1a-PE, anti-CD209-PE, anti-CD14-PE, anti-CD83-PE, anti-CD103-PE, anti-CX3CR1-PE, and anti-CCR7-PE (Beckman Coulter, Hialeah, FL, USA). The growth factor receptors were characterized by anti-GM-CSFRα-PE and anti-M-CSF R/CD115-PE (R&D Systems, USA) and isotype-matched control antibodies (BD PharMingen, San Diego, CA, USA). Fluorescence intensities were measured by FACS Calibur (BD Biosciences, Franklin Lakes, NJ, USA), and data were analyzed by the FlowJo software (Tree Star, Ashland, OR, USA). The human chemokines Mk, CXCL7, CCL20, and CXCL16 were ordered from PeproTech, UK; CXCL8 and CXCL1 are from Miltenyi Biotec.