White walled 96 well plate

For the propidium iodide staining, organoids were grown and incubated with varying concentrations of OA as described for the lipid droplet confocal assay. The organoids were then washed with Hank’s balanced salt solution (HBSS) (Gibco), and stained with Hoechst 1 μg/mL (Sigma-Aldrich) and propidium iodide 0.1 mg/mL (ThermoFisher) in HBSS at room temperature for 15 minutes. Organoids were imaged by an inverted Olympus IX53 epifluorescence microscope (Tokyo, Japan).
For the Caspase-Glo 3/7 assay (Promega, Madison, WI), organoids were grown on TC-treated 96-well plates (Greiner, Kremsmunster, Austria) and incubated with OA as was done for the propidium iodide staining. Organoids were washed and resuspended in HBSS and transferred to a white-walled 96-well plate (Greiner). The assay was performed according to the manufacturer’s protocol and luminescence was measured on a Tristar 2 luminometer (Berthold Technologies, Oak Ridge, TN).

Dissected retina or RPE-choroid were homogenized using a membrane disruptor (sonic dismembrator; Fisher Scientific, Hercules, CA, USA) at 30% power for 15 pulses in lysis buffer containing protease inhibitor (20 mM MOPS, pH 7.0; 2 mM EGTA; 5 mM EDTA; 0.1% Triton X-100; complete protease inhibitor tablet; Roche, Indianapolis, IN, USA). The homogenates were subsequently centrifuged at 10,000 g for 15 minutes at 4°C. The protein concentration of the supernatant was then measured using an assay kit (Dc Protein Assay; Bio-Rad Laboratories, Hercules, CA, USA). Caspase 8 activity was measured using a luminescent assay kit (Caspase-Glo 8 Assay; Promega, Madison, WI, USA) according to the manufacturer's instructions. Briefly, 100 μg of cytosolic protein was incubated with substrate in a white-walled 96-well plate (Greiner Bio-One, Monroe, NC, USA) at room temperature for 1 hour. The untreated retina and RPE samples and no-tissue blank wells served as controls. Luminescence was measured in a plate reader luminometer (Turner Biosystems, Sunnyvale, CA, USA).