Intestinal organoids were cultured in 24-well plates with a density of 200–300 organoids per well for 7–10 days. Secretion experiments were performed by incubating the organoids with test regents in treated solution (138 mM NaCl, 4.5 mM KCl, 4.2 mM NaHCO3, 1.2 mM NaH2PO4, 2.5 mM CaCl2, 1.2 mM MgCl2, and 10 mM HEPES supplemented with 0.1% (wt/vol) fatty acid-free BSA and 1% DPP-IV inhibitor, pH 7) for 2 h at 37°C. At the end of the incubation period, supernatant was collected, and the organoids were lysed in RIPA buffer supplemented with protease inhibitor and DPP-IV inhibitor. Incretins in supernatant and lysates were assayed using ELISA kit for GIP (#EZRMGIP-55K; Millipore) and total GLP-1 (#K1503PD-1; MSD). Secretion was first calculated as the percentage of secreted GLP-1 (supernatant) in total GLP1 (both supernatant and cell lysates) and then normalized to the basal secretion in WT organoids in parallel on the same day.
K1503pd 1
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The K1503PD-1 is a laboratory equipment product. It is a precision device designed for scientific and research applications. The core function of the K1503PD-1 is to perform specific tasks required in a laboratory setting.
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4 protocols using k1503pd 1
1
Intestinal Organoid Secretion Assay
2
Intestinal Organoid Secretion Assay
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Intestinal organoids were cultured in 24-well plates with a density of 200–300 organoids per well for 7–10 days. Secretion experiments were performed by incubating the organoids with test regents in treated solution (138 mM NaCl, 4.5 mM KCl, 4.2 mM NaHCO3, 1.2 mM NaH2PO4, 2.5 mM CaCl2, 1.2 mM MgCl2, and 10 mM HEPES supplemented with 0.1% (wt/vol) fatty acid-free BSA and 1% DPP-IV inhibitor, pH 7) for 2 h at 37°C. At the end of the incubation period, supernatant was collected, and the organoids were lysed in RIPA buffer supplemented with protease inhibitor and DPP-IV inhibitor. Incretins in supernatant and lysates were assayed using ELISA kit for GIP (#EZRMGIP-55K; Millipore) and total GLP-1 (#K1503PD-1; MSD). Secretion was first calculated as the percentage of secreted GLP-1 (supernatant) in total GLP1 (both supernatant and cell lysates) and then normalized to the basal secretion in WT organoids in parallel on the same day.
3
Oral Glucose and Oil Gavage Protocol
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Adult mice were given glucose (3 g/kg) via oral gavage after being deprived of food for 4–6 h. Tail blood was collected from tail at 0min and 10 min after glucose administration. Blood was collected and mixed with an inhibitor cocktail (Aprotinin, A6279, Sigma-Aldrich; DPP-IV inhibitor, DPP4–010, Millipore; 0.5M EDTA, AM9261, Invitrogen. 1:1:1, 10 mL mixture for each sample.). On another occasion, tail blood was collected at 0 min and 60 min after 100% corn oil (10 µL/g BW) via oral gavage after 4~6 h fasting. Total GIP (#EZRMGIP-55K; Millipore) and total GLP-1 (#K1503PD-1; MSD, Rockville, MD) measurements were measured in plasma samples.
4
Oral Glucose and Oil Gavage Protocol
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