Cd83 fitc

Non-specific binding sites on harvested cells were blocked with 20% inactivated AB-serum in PBS containing 0.1% BSA and 0.1% NaN₃ for 20 min at 4 °C. Then, cells were stained for 30 min at 4 °C with fluorescence-labelled antibodies: CD83-FITC, CD19-PECy7, CD80-APC647, CD14-APCCy7, HLA-DR-Brilliant Violet 421 (all Biolegend, San Diego, CA) CD86-PE, CD86-PECy7 (both BD Biosciences) and fixable viability dye eFluor506 (eBioscience, San Diego, CA, USA). Fluorescence was assessed by flow cytometry on a BD FACS Canto II and analyzed with FlowJo software.

Antibodies and other reagents used were fixable viability dye e-flour 450, Annexin V FITC (ebioscience); anti-human CD86-FITC, CD80-FITC, Propidium Iodide (PI) (BD Pharmingen, San Jose, CA); anti-human CD11c PE, HLA-DR PerCP, CD80-PE (Becton Dickinson, San Jose, CA); CD282-FITC, CD284-PE, Donkey anti-rabbit-FITC, CD86-PE, CD83-APC, CD11c-PE-Cy7, CD83-FITC, CD14-APC, and 7 aminoactinomycin D (7AAD (Biolegend, San Diego, CA). Data acquisition was on a Miltenyi MacsQuant flow cytometer (Miltenyi Biotech, San Diego, CA) on a log scale and analysis was performed with FlowJo 7.6.5 software (Tree Star, Ashland, OR).

The staining for cell surface proteins were performed following standard protocols by incubation of cells with pre-optimized concentrations of antibodies at 4°C for 20 min in FACS buffer (PBS with 2% FCS and 1 mM EDTA). Flow cytometry data were acquired on BD FACS Canto II cytometer and analyzed with FlowJo software version X (Tree Star). Unless otherwise stated all antibodies were supplied by BD Biosciences: CD25 APC (M-A251) or CD25 PECy7 (2A3); CD8 APC (SK1); CD4 V500 (RPA-T4), CD3 PerCPCy5.5 (SK7), CD38 PE (HIT2); CD80 PE (L307.4); CD83 FITC (HB15e); CD86 PECy7 (FUN-1), HLA-DR PerCP (L243); CCR7 PECy7 (G043H7; Biolegends). Appropriate isotypes were used as controls.