Leu15 gastrin 1

To generate gastric organoids (gastroids), mouse gastric mucosa from the corpus was isolated and incubated, with gentle rocking, at 4 °C for 2 h in 10 mM EDTA in Dulbecco’s phosphate-buffered saline (DPBS) pH 7.4 without Ca²⁺ and Mg²⁺. To dissociate the individual glands, the tissue was incubated and shaken in ice-cold buffer (43.3 mM sucrose (Thermo Fisher Scientific, Waltham, MA), and 54.9 mM D-sorbitol, (Sigma-Aldrich, St. Louis, MO), in DPBS). Glands were centrifuged at 150×g for 5 min at 4 °C, and the pellet was re-suspended in Matrigel^® (Corning, Corning, NY) and added to an eight-well Lab-Tek chamber with coverglass (Thermo Fisher Scientific, Waltham, MA). The Matrigel^® was allowed to polymerize at 37 °C. Then medium was added to the wells and replaced every 4 days and cultured in a 5% CO₂ incubator at 37 °C. Gastroid media consisted of advanced DMEM/F12 supplemented with 2 mM GlutaMax^® (Thermo Fisher Scientific, Waltham, MA), 10 mM HEPES (Research Organics, Cleveland, OH), 100 U/mL penicillin/100 μg/mL streptomycin, 1 × N2 and 1 × B27 supplements (Thermo Fisher Scientific, Waltham, MA), Wnt3a-conditioned medium (50%), R-spondin-conditioned medium (10%), Noggin-conditioned medium (10%), [Leu15]-Gastrin I (10 nM, Sigma-Aldrich, St. Louis, MO), and epithelial growth factor (50 ng/mL, PeproTech, Rocky Hill, NJ) [31 (link)–33 (link)].

Fetal bovine serum (FBS), DMEM, Minimum Essential Medium Eagle (MEM), CDCA, n-acetylcysteine (NAC), [Leu]15-Gastrin 1, NT peptide and β-actin antibody were from Sigma-Aldrich (St. Louis, MO). GW4064 was from Tocris (Minneapolis, MN). The phospho ERK1/2 and ERK1/2 antibodies were from Cell signaling (Danvers, MA). FXR and NTR1 antibodies were from Santa Cruz (Dallas, TX). Noggin-conditioned medium was purchased from U-Protein Express BV (Netherlands). Advanced DMEM/F12 medium, OptiMEM Reduced Serum Medium, growth factor–reduced Matrigel, B-27 Supplement, N-2 Supplement, HEPES, GlutaMAX and Zeocin were from ThermoFisher (Grand Island, NY). Mouse EGF was from PeproTech (Rocky Hill, NJ).