Chir 99021 chir

Small intestines of 7–10 weeks old Cd44^fl/fl;VillinCreER^T2 mice were washed with PBS and opened longitudinally. Intestines were placed villi facing up. Villi were gently removed using a glass slide and the intestines were washed in PBS until no cell debris remained. The washed intestines were incubated with 2 mM EDTA for 30 min at 4 °C. Crypts were mechanically released from the surrounding tissue by shaking. The freed crypts were pelleted and passed through a 70 μm strainer. Isolated crypts were resuspended in Matrigel (Corning) and cultured in 500 μl complete IntestiCult™ Organoid Growth Medium (STEMCELL Technologies) based on the previously described standard ENR medium (Adv. DMEM F12 (Sigma), 50 ng/ml EGF (Peprotech), 100 ng/ml Noggin (Peprotech), 3 µm CHIR99021 (CHIR) (Selleckchem), B27 (Gibco), N2 (Gibco), 1 mM N-Acetyl cysteine (Sigma)) [16 (link)] in 24-well plates. 4-hydroxytamoxifen (4-OHT) (1 mM) (Sigma) or ethanol was added for 48 hours. Organoids were split by a mechanical breakup and dispersed crypts were reseeded every fifth day in a 1:2 ratio for 2 passages. Organoids were treated with CHIR (15 µM) or DMSO in IntestiCult™ Organoid Growth Medium (STEMCELL Technologies) or in ENR supplemented with 50% Wnt3a CM and 20% R-Spondin CM or 5% R-Spondin CM only.