IFN-β Bioassay for Peptide-dsDNA Complexes

IFN-β bioassay was carried out similarly to prior work^{55 (link)}. Supernatants were assayed for type I IFN production (IFN-β) using ISRE-L929 IFN reporter cells (Bio-ELISA system with IFN-β receptor coupled to a luciferase promoter). ISRE-L929 cells were grown to confluency in RPMI + BGS + Pen/Strep + l-glutamine and plated to a final concentration of 10⁵ cells per well in a 96-well microplate. Fifty microliters of supernatant was incubated with ISRE-L929 cells for 7 h at 37 °C. An eight-point standard curve with twofold serial dilutions of known IFN-β concentrations (R&D Systems) was incubated with ISRE-L929 cells at the same time. ISRE-L929 cells are lysed overnight at −80 °C in Passive Lysis Buffer (Promega). Luciferase production in ISRE-L929 is quantified using a Luciferase Assay System (Promega) with a luminometer plate reader (Biotek). Luciferase production was converted to IFN-β production using the calibrated IFN-β standard curve. Peptide-dsDNA complexes that induce a TLR9-specific response produce a significantly larger amount of IFN-β from WT stimulation compared to TLR9KO stimulation (unpaired Student’s t-test).