Anti cd86 apc

Cells were washed with PBS and stained at room temperature using a 1:10 dilution of the indicated antibodies in 100 µL PBS for 10 min. Cells were washed once more to remove antibody excess and flow cytometry data was subsequently acquired with FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA). Live cells were gated based on forward and side scatter plots. Isotype staining’s were performed as control conditions. Anti-CTLA-4 PE extracellular antibody, anti-Gal-9-PE and their corresponding isotype controls were purchased from Biolegend, San Diego, CA, USA. The rest of the antibodies (anti-CD276-PE, anti-CD80-PE, anti-CD86-APC, anti-CD273-PE, anti-CD274-APC, anti-HLA-E-APC, anti-CD112-PE, anti-CD66abce-APC, anti-CD155-PE, anti-CD19-APC or FITC, anti-PD1-PE, anti-TIM3-APC, anti-NKG2A-FITC, CD56-APC, CD96-APC, TIGIT-PE, CD34-APC, CD271-PE and corresponding isotype controls) were obtained from Miltenyi Biotec, Bergisch Gladbach, Germany. CD19-CAR and CD276-CAR expression on NK-92 cells was determined by CD271 and CD34 marker gene expression, respectively, whereas luciferase expression on target cells was correlated with GFP for Nalm-6 GFP/Luc and CD19 marker expression for U-937 CD19tag/Luc.

Purity and phenotype of mDCs and pDCs after CliniMACS isolation were determined by flow cytometry with a FACSVerse (BD Biosciences, San Jose, CA, USA) or MACS Quant (Miltenyi Biotec). The following primary monoclonal antibodies and the appropriate isotype or fluorescence minus one controls were used: anti-CD1c-Viobright FITC, anti-BDCA-2-PE, anti-CD20-PE-Vio770, anti-CD123-APC, anti-CD45-APC-Vio770, anti-CD14-VioGreen, anti-FcεRI-VioBlue, anti-CD14-FITC, anti-CD15-PE, anti-CD56-APC, anti-CD3-BioBlue, anti-HLA-ABC-APC, anti-HLA-DR,DP,DQ-APC, anti-CCR7-APC, anti-CD80-APC, anti-CD83-APC and anti-CD86-APC (all Miltenyi Biotec).