Intestinal Organoid Secretion Assay

Small intestinal organoid generation from mouse jejunum (section taken 8–13 cm from the gastric pylorus), maintenance, and two-dimensional (2D) culture were carried out as previously described [50 (link)]. GIP secretion experiments were performed using 2D organoid-derived cultures, 18–24 h after seeding cells onto 48-well plates. Cells were washed three times in warm saline buffer (138 mM NaCl, 4.5 mM KCl, 4.2 mM NaHCO₃, 1.2 mM NaH₂PO₄, 2.6 mM CaCl₂, 1.2 mM MgCl₂, 10 mM HEPES; adjusted to pH 7.4 with 1 M NaOH, and supplemented on the day of experiment with 1 mM glucose and 0.1% fatty acid-free bovine serum albumin) before incubation in saline buffer for 30 min at 37 °C. The buffer was then completely removed before test reagents, dissolved in 150 µL saline buffer, were added to the organoid cultures. The test reagents used were 100 ng/mL LPS (Sigma-Aldrich, St. Louis, MO, USA), 100 µg/mL recombinant human IL-1Ra (PeproTech, Rocky Hill, NJ, USA), 10 ng/mL recombinant human MCP-1 (Peprotech), or 10 µM forskolin plus 10 µM 3-isobutyl-1-methylxanthine (IBMX) plus 10 mM glucose (all Sigma-Aldrich, St. Louis, MO, USA). After incubation for 2 h at 37 °C, supernatants were removed and centrifuged at 2000× g for 5 min at 4 °C, transferred to a fresh tube, and snap-frozen on dry ice.