Cd86 af700

For immunoflourescent staining of tissue sections the following primary antibodies were employed: CD68, 1 µg/ml (clone: PGM-1, Dako Cytomation, Hamburg, Germany); CD14, 1 µg/ml (clone: TÜK4, Dako); CD86, 10 µg/ml (clone: FUN-1, BD Pharmingen, Heidelberg, Germany). Isotype- and concentration-matched control antibodies (Dako) served as negative controls.
For flow cytometric analysis the following mAb were purchased from BD Bioscience (Heidelberg, Germany): CD3 V450, CD14 FITC, CD33 PE-Cy7, CD86 AF700, HLA-DR V500, CD117 APC.

Discrimination of viable cells was based on staining with the Live/Dead Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Following Live/Dead staining, pellets were resuspended in Brilliant Staining Buffer (BD Biosciences), treated with the FcR blocking reagent (Miltenyi Biotec), and stained with a multicolor antibody panel consisting of CD11c-FITC, CD40-BV711, CD80-BV480, CD83-BV605, CD86-AF700, PD-L1-BV421, and HLA-DR-BV786 (BD Biosciences). Samples were acquired on a FACSAria III flow cytometer (BD Biosciences) with automatic compensation based on single-stained mDC and analyzed in FlowLogic (Inivai Technologies). At least 25,000 CD11c+ events were recorded. The gating strategy was based on size/complexity (FSC/SSC), singlet discrimination (FSC-area/FSC-height), viability (APC-Cy7-negative), and CD11c+ (FITC) as illustrated in Figure 2. The median fluorescent intensity (MFI) was measured on CD11c+ cells, to exclude them possibly from ASCs that are CD11c-negative. The MFI was normalized to the intensity of the mDC phenotype of the given donor.

Single cell suspensions were incubated for 5 min with 5% human serum (Sanquin Blood Bank) in PBS to block non-specific Fc-receptor binding, washed in PBS/0.1% BSA (Merck, Darmstadt, Germany) and stained with monoclonal antibodies against cell surface markers CD11b-PE, CD1a-BV605, CD80-FITC, CD86-AF700 (all BD BioSciences, Vianen, The Netherlands), CD14-FITC, and CD163-AF647 (BioLegend, San Diego, CA, USA) for 30 min at 4°C. Cells were washed twice in PBS/0.1% BSA and acquisition was performed using a BD FACSLyric™ Flow Cytometer (BD Biosciences). Data was analyzed using FlowJo v10 software.