Migg1 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).

Cells were washed and resuspended in staining buffer. Fluorochrome-conjugated monoclonal antibodies were added to cells and incubated for 30 min in the dark. An hUCB-MSC surface marker analysis with flow cytometry was performed using the following monoclonal antibodies: CD105, CD73, CD90, CD45, CD34, CD14, CD11b, CD19, HLA-DR, positive isotype control cocktail (mIgG1 FITC, mIgG1 PErCP-Cy5.5, and mIgG1 APC), and negative isotype control cocktail (mIgG1 PE and mIgG2a PE) purchased from BD Biosciences (San Jose, CA, USA). Phenotyping of macrophages with flow cytometry was performed using the antibodies of M2 macrophage differentiation markers (CD163, BD Biosciences; CD206, Abcam, Cambridge, UK) and M1 macrophage differentiation markers (CD80; Abcam). After staining of primary antibody, cells were washed and resuspended in 150 μL of the appropriate secondary antibody (Alexa Fluor® 488-conjugated donkey anti-rat, Alexa Fluor® 647-conjugated donkey anti-rabbit; Abcam). Data were acquired using BD LSRFortessa flow cytometers and analyzed with BD FACSDiva software (BD Biosciences, Franklin Lakes, NJ, USA).

Fluorescent conjugates targeting human (h)RANK (clone 80704, R&D Systems, Minneapolis, MN, USA), CD33, CD117 (both Biolegend; San Diego, CA, USA), CD13 (DAKO, Santa Clara, CA, USA), CD34, 7AAD (both BD Biosciences, Heidelberg, Germany) were used. RANK expression was detected by flow cytometry using mIgG1-PE (BD Biosciences) as isotype control. Analysis was performed on a FACSCanto^TM II or a FACS LSRFortessa^TM instrument (both BD Biosciences). Specific fluorescence indices (SFI) were calculated as follows: “median fluorescence intensity obtained with specific monoclonal antibody (mAb)” divided by “median fluorescence intensity obtained with isotype control”. Surface positivity was defined as SFI ≥ 1.5.