2.5 Luciferase assay

To prepare lipoplexes for transfection, reporter firefly luciferase (Luc)-encoding RNA was incubated with liposomes following proprietary inhouse protocols. RNA was provided as a HEPES-buffered solution and diluted with 1.5 M NaCl and water to a concentration of 150 mM NaCl and an RNA concentration of 0.1 mg/mL, which results in a molar charge ratio of 0.65:1.00 (positive:negative) upon incubation with liposomes. The charge ratio was calculated from the number of positive charges present in lipid head groups and the number of negative charges derived from the number of RNA nucleotides (330 Da per nucleotide was assumed). Liposomes were in-house generated containing (R)-N,N,N-trimethyl-2-3-dioleyloxy-1-propanammonium chloride (DOTMA) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in a molar ratio of 2:1 (DOTMA:DOPE). Lipoplexes were then mixed with hiDCs, and the transfected cells were seeded in 96-well plates with a density of 5 × 10^4 cells and 0.5 µg RNA per well, in media containing 0.4 ng/μL IL4 and GM-CSF. Luciferase assays were conducted at 6, 24, 48 and 72 h after transfection using the Promega BrightGlo Kit according to manufacturer’s instructions.