Mouse colon organoid culture and immunofluorescence staining

Mouse colon organoid isolation was performed according to the manufacturer's protocols. In brief, the colon was harvested and luminal contents were flushed away by cold PBS in a 10 cm dish. The colon was then transferred to a biosafety cabinet and cut into 2-5 mm pieces. Colon pieces were washed thoroughly in cold PBS, and crypts were digested using Gentle Cell Disassociation Reagent (StemCell Technologies) on a rocking platform for 20 min. The supernatant was removed, and colon pieces were resuspended in cold PBS with 0.1% BSA. The new supernatant was then passed through a 70 μm strainer and centrifuged at 4 ºC 290×g for 5 min to obtain crypts. Isolated crypts were then resuspended in DMEM/F12 with 15 mM HEPES (StemCell Technologies) and counted under a microscope. An equal volume of IntestiCult Organoid Growth Medium (StemCell Technologies) and Matrigel (Corning) were mixed and the suspension was transferred into a pre-warmed 24-well-plate to form domes in the middle of each well. Complete IntestiCult Organoid Growth Medium was added above the domes, and crypts were cultured at 37 ºC and 5% CO₂.

For the PCNA staining, colon organoids were fixed by paraformaldehyde and incubated with anti-PCNA antibody (CST) at 4 ºC overnight and goat-anti-rabbit IgG (FITC Conjugate, Beyotime) at room temperature for 1 h. Organoids were then observed under a fluorescence microscope (IX73, Olympus).