Adf 12

Intestinal crypts were isolated from the proximal part of the small intestine of 6–10 weeks Mask1 fl/fl Mask2 fl/fl mice (generated from EUCOMM ES cells for this study) as follows. The whole gut was harvested and washed in cold DPBS (#14190250, Gibco). The most proximal 5 cm were cut open, and the villi scraped off with a coverslip. The remaining tissue was cut in 0.5 cm pieces, washed several times by pipetting up and down in 8 mL fresh DPBS, and incubated in DPBS + 2 mM EDTA for 30 min at 4°C. Crypts were then mechanically extracted by vigorous shaking in DPBS, and filtered through a 70 µM Nylon cell strainer (#352350, Falcon). After several low speed washes in ADF-12 (#12634–010, Gibco), isolated crypts were resuspended and plated in Matrigel (#354230, Corning). Organoids were cultured in IntestiCult medium (#06005, Stemcell technologies) supplemented with Primocin antibiotic (1:50, ant-pm-2, Invivogen), either in 24-well plates for maintenance, or in 8-well chambers (#80827, Ibidi) for Ad-Cre-GFP infection and immunostaining. Passages were performed by resuspending Matrigel-embedded organoids in cold DPBS and transferring them to a Falcon tube using a 2 mL syringe with a 27 G ½ needle (BD Microlance #300635) to break them up. After two low speed washes in ADF-12, organoids were resuspended and plated in Matrigel.

Intestinal crypts were isolated from 6‐ to 10‐week‐old C57B/l6 or transgenic mice, following the published protocol from Mahe et al (2013). Briefly, the whole gut was harvested and washed in cold DPBS (Gibco, 14190250). The most proximal 5 cm were cut open, and the villi were removed with a coverslip. The remaining tissue was washed and incubated in 2 mM EDTA for 30 min at 4°C. Two crypt fractions were then mechanically extracted, the first one being filtered through a 70 μM cell strainer before pooling both fractions together. After several low speed washes in ADF‐12 (Gibco, 12634‐010), isolated crypts were resuspended and plated in Matrigel (Corning, 354230). Organoids were cultured in IntestiCult media (Stemcell technologies, 06005) complemented with Primocin antibiotic (Invitrogen, ant‐pm‐05). Organoids were cultured in 24‐well plates for maintaining the cultures and then cultured in 8‐well chambers for drug incubations and immunostaining (Ibidi, 80827). The Porcupine inhibitor LGK974 (Selleck chemicals, S7143) was used at 5 μM for 24 h. For Src inhibition experiments, organoids were treated with 100 nM of Dasatinib (Selleck chemicals, S1021) or 100 nM eCF506 for a period of 4 h. Organoid microscopy was performed with either a Leica SP5 or a Leica SP8 laser‐scanning confocal microscope.