In vitro transfection, luciferase assay, and cell viability assay

HEK293 cells were seeded in white 96-well plates at a density of 12 × 10³ cells per well in 100 µL EMEM medium (10% FBS) the day before transfection and incubated at 37 °C 5% CO₂. FLuc mRNA-loaded LNPs were diluted so that 8, 16, 24, and 32 µL volumes contained 25, 50, 100, and 200 ng mRNA FLuc and HEK293 cells were transfected using triplicates for each of the 4 doses 24 h after seeding. After a further 24 h for transfection, 100 µL of One-Glo substrate was added directly to the wells to detect luciferase expression based on luminescence in Cytation 5 luminometer plate reader. A toxicity assay was performed 24 h after transfection in a separate plate, where transfected cells were incubated with pre-warmed Presto Blue HS reagent (10% v/v) for 15 min at 37 °C. Microplates were immediately introduced into the Cytation 5 to read Fluorescence (Ex540/Em590).