Crypt isolation and organoid culture.

The intestinal organoids were derived from the small intestines as reported (24), with slight modifications. In brief, 10 cm small intestines were dissected and opened longitudinally to remove luminal contents. The intestine was cut into 5 mm pieces and incubated with 4 mM EDTA in PBS for 30 minutes at 4°C without shaking. Crypts were dissociated from villi by pipetting and filtered through a 70 μm strainer (BIOLGIX), followed by centrifugation (4°C, 200g, for 5 minutes) and washing. The purified crypts were resuspended in Matrigel (Corning, 356231) and seeded onto a glass-bottom dish, then cultured in IntestiCult Organoid Growth Medium (StemCell Technologies, 06005). Organoid growth medium was refreshed every 2–3 days. For the PI-traced organoid cell death assay, TNF-α (50 ng/mL), SM-164 (50 nM), and Z-VAD-FMK (50 μM) were added into organoid growth medium for 8 hours at day 7. Then organoids were stained with 1 μg/mL PI, and images were taken using a confocal microscope (FV3000, Olympus).