Intesticult ogm mouse kit

Cut mouse colon samples into 4–5 mm and then were incubated in 20 mM EDTA in PBS for 30 min at 4°C. For mouse organoids, 200 crypts per well in 24‐well plates were suspended in 50 μL Matrigel composed of 25% IntestiCult OGM Mouse Kit (Cat. 06005, Stemcell) and 75% growth factor‐reduced Matrigel (Cat. 354230, Corning). The Matrigel suspension was placed at 37°C for 30 min to fix. Then 500 μL fresh complete medium with 10 mM Y‐27632 (Cat. S1049, Sellceck, China) was added to the Matrigel for the first 48–72 h of culture. Day 3, colonoids were treated with L‐arginine (0.4 mM; Cat. PHR1106, Sigma, USA) or PBS. The complete medium was replaced every 2 days. Images were taken on Day 3, Day 5, and Day 7. We captured the images of colon organoids in each well using microscope (Carl Zeiss). Then the diameter and buds of colon organoids was analyzed using the Zen image program (Carl Zeiss).

Mouse intestinal stem cell (ISC) organoids were isolated and harvested from CD-1 mice (courtesy of the Posfai Laboratory, Princeton University) according to Stemcell Technologies^TM’s mouse intestinal crypt isolation protocol, then maintained in culture using reagents and protocol from the IntestiCult OGM Mouse Kit (Stemcell Technologies, Vancouver, Canada). Intestinal organoids were disassociated and resuspended into Corning®Matrigel®Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-free, LDEV-free, 10 mL (Corning) at a 1:1 ratio with organoid growth medium. 27.5μL of the mixture was deposited onto each of the rectangular stencils and allowed to solidify for 15 min at 37 °C before the gel was covered with organoid growth medium and cultured for 72 h.