Optimization of assay for detection of innate immune stimulation of PBMCs by siRNA. Supernatant from PBMCs treated with DOTAP complexed siRNAs for 24 hours was applied to Huh7-Luc cells. Luciferase expression was assayed 24 hours after treatment with supernatant. Each point represents the mean of triplicate samples. Error bars indicate standard deviation. (A) The indicated densities (cells/100 μL) of PBMCs were stimulated with 40 nM indicated siRNAs. (B)-(E) Dose response curves performed using PBMCs at a density of 50,000/100 μL in the presence or absence of chloroquine at the indicated concentrations.