Retinal Explant Culture and Treatments

Retinal explants obtained from newborn rd1 and wt mice were cultured using a protocol from previously described methods^{10 (link), 19 (link), 62 (link)}. Briefly, both eyes were enucleated and retinas were carefully peeled away from the retinal pigment epithelium, and radial cuts were made to flatten the retina. The flattened retina was transferred to the membrane of a Millicell insert (Millipore, PICM03050) with the photoreceptors facing down. The insert was placed into the wells of a 6-well plate (Costar 3516, Corning), each contained 1300 μl of retinal explant media, which was replaced every 2 days, and was maintained in a 37 °C incubator with 5% CO₂ for 10 days. The retinal explant basal medium was serum-free and made from Neurobasal A, DMEM/F12 medium, and N2/B27 supplements (Life Technologies, USA). Cultured explants were either exposed to DZNep (Cayman Chemical, 13828, Ann Arbor, USA) treatment (0.1 μM, 0.5 μM, 1 μM, 1.5 μM), LY294002 (Selleckchem, S1105, Houston, TX, USA) treatment (20 μM), or kept as PBS-treated controls.