Interleukin 6 (il 6)

Supernatants from stimulated cultures were collected at 6 hours and stored at −80°C until analysis. The ELISA analysis was performed using commercially available kits for human IL-1β, interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α) (BD Biosciences).

Human PBMCs were isolated from heparinized blood of healthy volunteers upon informed consent and approval by the local ethical committee (admission number: S-716/2017) by standard Ficoll-Hypaque density gradient centrifugation (Ficoll 1.078 g/mL). PBMCs were resuspended in RPMI 1640 supplemented with 2% heat-inactivated human serum. For transfection experiments, all RNAs were encapsulated with DOTAP at a ratio of 3 µL DOTAP per 1 µg of RNA in serum-free medium according to the manufacturer's protocol. Unless otherwise indicated, cells were stimulated with RNA preparations at a final concentration of 500 ng/mL, and transfection was performed at a density of 2 × 10⁵ PBMCs per well in a 96-well flat bottom plate at a final volume of 100 µL. Cells were incubated overnight in a humidified 5% CO₂ atmosphere at 37°C. For cytokine measurement, levels of TNF, IL-6 (BD), and IFN-α (eBioscience) were determined in cell-free supernatants by ELISA.