Anti cd83 fitc clone hb15e

moDCs were stained with the fixable viability dye eFluor 780 (eBiosciences, San Diego, CA, USA) for 20 min, followed by staining with anti-HLA-DR APC (clone G46-6, BD Biosciences), anti-CD83 FITC (clone HB15e, BD Biosciences), anti-CD86 PE-CF594 (clone 2331, BD Biosciences), anti-CD1c PerCP-eFluor™ 710 (clone L161, Thermo Fisher Scientific), and anti-CD45 BV510 (Clone HI30, BD Biosciences). Cytokines (IL-1β, IL-6, IL-8, IL-10, IL-12p70, and TNF) were measured in supernatants using the BD™ Human Inflammatory Cytokine Cytometric Bead Array (CBA), following the manufacturer’s instructions. Flow cytometry data were acquired using the FACS Aria III (BD, Franklin Lakes, USA) and analyzed with Kaluza, version 2.1 (Beckman Coulter Life Sciences, Indianapolis, USA).

DCs were transduced with different Ad5 vectors at a ratio of 5000 virus particles (measured using the Quant-it PicoGreen assay kit, Invitrogen) of Ad5 virus per cell for 1 hr at 37°C. Cells were harvested and resuspended at a final concentration of 10⁵ cells/mL in culture medium (RPMI-1640 supplemented with 5% pooled human AB serum (Sigma Aldrich, UK), 1% penicillin, 1% streptomycin and 1% L-glutamine (PAA Laboratories)). Cells were incubated overnight then matured the following day with bacterial lipopolysaccharide (LPS, 1 µg/ml, Sigma Aldrich) and IFNγ (1000 U/ml, Miltenyi Biotec) for a further 24 hrs.
To assess proteasomal degradation of targeted vaccine genes, DCs were transduced with the different Ad5 vectors for 24 hrs, and following maturation were cultured in the presence or absence of a 10 µM of a proteasomal inhibitor, MG132 (Sigma-Aldrich, UK) for a further 24 hrs.
To assess the impact of transduction on the maturation of DCs, DCs were transduced with the different Ad5 vectors for 24 hrs, matured with LPS and IFNγ for a further 24 hrs and then surface stained for anti-CD80-PE (clone 2D10, BioLegend), anti-CD83-FITC (clone HB15e, BD UK), anti-CD86-APC (clone IT2.2, BioLegend), anti-HLA-DR-PECy5 (clone G46-6, BD, UK) and anti-PDL1-FITC (clone MIH2, AbD Serotec).