Measuring Type I IFN Levels in Mouse Retina

Mouse IFN-α (42115-1, pbl Assay Science) and IFN-β (42410, pbl Assay Science) ELISA kits were used to measure the level of IFN-β in mouse retinal and primary mREC culture medium. Mice were euthanized by CO₂ asphyxiation. The retina was freshly isolated and sonicated in a sample buffer provided in the ELISA kit with 1% phosphatase and 1% protease inhibitors. The mREC culture medium was collected 4 days after cGAMP (20 μg/mL; vac-nacga23, Invivogen) and STING inhibitor (2 μg/mL; inh-h151, Invivogen) treatment. Following the kit instructions, the absorbance of each sample was measured by a microplate reader (BioTek Synergy H1 Hybrid reader) at 450 nm. Lastly, the IFN titer of the samples was determined by plotting the optical densities using a 4-parameter fit for the standard curve in GraphPad Prism 9 (version 9.3.1). The retinal explants from WT, STING-KO, and STING^GT mice were cultured in presence or absence of LG or HG as explained above and in a previous report (55 (link)). The spent media from the cultures were analyzed by ELISA for HMGB1 (Novus Biologicals, NBP2-62767) and IL-1β (Invitrogen, BMS6002) using commercially available kits and following the manufacturer’s guidelines.