Advanced dulbecco s modified eagle medium f12

Crypts were isolated from the small intestine of male WT mice aged 7–11 weeks or small intestinal tumors of female APC^min mice aged 21 weeks as previously described.²⁴ (link) One mouse was used for each isolation. The crypts were embedded in Matrigel (Corning, Corning, NY). Advanced Dulbecco’s modified Eagle medium/F12 (Thermo Fisher Scientific) containing 50 ng/mL mouse Epidermal Growth Factor (mEGF) (Peprotech, Rocky Hill, NJ), 100 ng/mL mouse Noggin (mNoggin) (R&D Systems), 100 U/mL penicillin/streptomycin (Nacalai, Kyoto, Japan), 10 mmol/L HEPES (Nacalai), 2 mmol/L GlutaMAX-1 (Thermo Fisher Scientific), 1 mmol/L N-acetylcysteine (Sigma, St. Louis, MO), 1× N2 supplement (Thermo Fisher Scientific), 1× B27 supplement (Thermo Fisher Scientific), and 10 μmol/L Y-27632 (Wako, Osaka, Japan) were added to each well (complete medium). For WT organoids, 500 ng/mL mouse Rspondin1 (mRspondin1) (R&D Systems) was added. Organoids from APC^min mice were passaged at least once before co-culture with IECs.

Tissues were washed in phosphate-buffered saline (PBS), and mucosa was isolated by removal of outer muscle layers using surgical scissors. Mucosae were cut into 1-cm pieces and washed with PBS until the supernatant was clear. Small intestine and colonic crypts were isolated using modified existing methods.⁴ (link)^,6 (link) Specimens were incubated at 4°C in 8 mmol/L EDTA and 1 mmol/L dithiothreitol for 30 minutes to 1 hour, with gentle agitation. Supernatant was removed and replaced with fresh PBS. Specimens were vortexed to release crypts. Crypts were collected into 15-mL tubes and the process was repeated to collect 4–6 fractions. Fractions were centrifuged at 100 × g to pellet crypts. SI crypts subsequently were filtered with 100-um mesh to remove villus domains and epithelial debris. Isolated crypts were resuspended in basic medium containing Advanced Dulbecco's Modified Eagle Medium/F12, 1× Glutamax, 10 mmol/L HEPES buffer, and 1× penicillin/streptomycin (all ThermoFisher Scientific, Waltham, MA) for spheroid cultures, or resuspended in PBS for DNA lysis.

The entire small intestine from 8- to 12-week-old male and female mice was collected, and then the duodenum was removed. The remaining tissue was flushed with cold PBS, cleaned of the mesentery, and opened lengthwise. EDTA-based (15575020; Invitrogen) dissociation was performed as previously described.⁶⁹ (link) After EDTA dissociation, crypts were resuspended in Advanced Dulbecco’s modified Eagle medium/F12 (12634-010; Gibco) supplemented with GlutaMAX (35050061; Thermo Fisher Scientific), HEPES (15-630-080; Thermo Fisher Scientific), antibiotic–antimycotic (15240062; Fisher Scientific), 10% fetal bovine serum (900-108; Gemini Bio-Products), B27 (17504044; Thermo Fisher Scientific), N-2 supplement (17502048; Fisher Scientific), N-acetyl-L-cysteine (A9165; Sigma-Aldrich), EGF (PMG8043; Thermo Fisher Scientific), Noggin (250-38; PeproTech), Rspo1 (3474-RS-050; R&D Systems), and CHIR (SML1046; Sigma-Aldrich). Crypts were counted under a microscope, and then 200 crypts were seeded into 20 uL GFR Matrigel (356231; Corning) and plated in a prewarmed 24-well plate as domes. dmPGE2 and/or EP4i treatment began 24 hours after plating, and media was replaced every 2 days. Organoid size and number data were collected on day 6 after plating.