ISRE reporter assay

Macrophage-secreted type I IFN levels were determined using a L929 cells stably expressing a luciferase reporter gene under the regulation of type I IFN signaling pathway (L929 ISRE cells). At the indicated times post-infection, macrophage cell culture media was harvested and stored at -80°C. On the day prior to the assay, 5x10⁴ L929 ISRE cells were added to each well of a white 96-well flat-bottomed plate and incubated at 37°C/5%CO₂ overnight. On the day of the bioassay, a 1:5 dilution of media from infected macrophages was added to each well of L929 ISRE cells, then incubated for 5h. Cells were washed with 1X PBS, lysed in reporter lysis buffer, then 30 μl of Luciferase Assay System solution (Promega) added and luminescence read immediately using a Cytation5 plate reader.