Orion l

Luciferase promoter assays were performed using the Dual-Luciferase Reporter System (Promega) and assessed using the microplatte illuminometer ORIONL (Berthold). The transfection efficiency was controlled using the corresponding empty control vector (pGL3.1: 300 ng firefly luciferase respectively). The obtained data were normalized to the corresponding control vectors.

Confluent B16 cells were transfected in a 96-well plate with 0.1 μg of the respective reporter plasmid for promoter (binding) activity of IFN-β, IRF3, NFκB, AP-1, STAT3, ISRE or p53, respectively, 0.2 μg of the adjuvant plasmids (or a GFP-encoding control plasmid), and 0.3 μg polyethylenimine in DMEM supplemented with 10% FCS and 1% penicillin/streptomycin. After 48 hours incubation at 37°C and 5% CO₂, the cells were lysed with 100 μl Glo-lysis buffer, 25 μl Bright-Glo Luciferase substrate (both Promega) was added, and the signal was measured on a microplate luminometer (Orion L, Titertek Berthold).

Antigen-specific antibody responses were analysed by an ELISA as described in our previous study [21 (link)]. In short, ELISA plates were coated with 5x10⁵ PFU heat-inactivated influenza virus in 100 μl carbonate buffer (50 mM carbonate/bicarbonate, pH 9.6) per well over night at 4°C. After blocking free binding sites with 5% skimmed milk in PBS-T (PBS containing 0.05% Tween-20) for one hour at RT, serum dilutions in 2% skimmed milk in PBS-T were added to the wells for one hour at RT. After three washing steps each with 200 μl PBS-T, specifically bound antibodies were detected by incubation with HRP-coupled antibodies against murine IgG1 and IgG2a, respectively, for one hour at RT (all 1:1000 dilution; clone X56 and clone R19-15; BD Biosciences). Subsequently, the plates were washed seven times with PBS-T and after the addition of ECL solution, the signal was measured on a microplate luminometer (Orion L, Titertek Berthold).