One glo d luciferin substrate

PBMCs were harvested from mouse and human blood and plated at a density of 125,000 cells per well in a clear 96-well plate and immediately transfected. 50 μL of pre-warmed transfection medium and 50 μL of the polyplex solution (containing 100 ng of saRNA) were added to each well for 4 h. Transfection media were then removed, and the cells were cultured with cDMEM supplemented with 0.1 mM β-mercaptoethanol (Sigma-Aldrich, UK) for 4, 24, and 48 h. Positive control wells were stimulated with 20 mM TNF-α in cDMEM. At each time point, 50 μL of the culture media was removed and frozen at −80°C until further cytokine analysis. Then, 50 μL of ONE-Glo d-luciferin substrate (Promega, UK) was added and mixed well by pipetting. The total volume from each well was then transferred to a white 96-well plate (Costar) for analysis and quantified on a FLUOstar Omega plate reader (BMG Labtech, UK). The cytokine response in each well was quantified with a custom mouse or human 25-plex ProcartaPlex immunoassay (Thermo Fisher Scientific, UK) on a Bio-Plex 200 system (Bio-Rad) according to the manufacturer’s instructions.