Total bile acids test wako

Fecal pellets were weight, homogenized in ethanol. Total bile acids in feces was extracted by hot ethanol method, and analyzed using Total bile acids-Test wako (FUJIFILM Wako Pure Chemical) according to the manufacturer’s instructions.

Feces were dried for 6 hour at 90°C. For fecal total bile acid measurement, dried feces (approximately 0.2 g) were homogenized in distilled water (2 mL) and then shaken at 37°C for 15 minutes after the addition of t‐butanol (2 mL). After centrifugation twice, the supernatants were pooled and transferred to new tubes by each sample, which were then ready for measurement. Total bile acid values in the samples were measured using an enzymatic colorimetric assay kit (Total Bile Acids Test Wako; Wako Pure Chemical Industries, Ltd, Osaka, Japan). For fecal lipid measurement, dried feces (approximately 1.0 g) was homogenized in a 4 mL of hexane‐isopropanol (3:2) solution and then shaken for 10 minutes at room temperature. The supernatants from two centrifugation steps were pooled, evaporated to dryness, and solidified at 50°C in a nitrogen atmosphere to form a dried residue. After the residues were dissolved in a 1 mL of dioxane‐polyoxyethylene (1:1) solution, the samples were prepared for measurement. Total cholesterol and TG values in the samples were measured using the enzymatic colorimetric assay kits, Cholesterol‐E Test Wako and Triglyceride‐E Test Wako (Wako Pure Chemical Industries, Ltd), respectively. Fecal total bile acid and lipid values were normalized to each dried fecal weight.

To assess the ability of organoids to secrete albumin, culture medium was collected 48 hours after the last medium change. Albumin in the collected supernatant was quantified by Western blotting using an antibody against human serum albumin (ab8940; Abcam). To assess the ability of organoids to secrete bile acid, culture medium was collected 48 hours after the last medium change. Bile acid in the collected supernatant was quantified using Total Bile Acids Test Wako (Wako Pure Chemical Industries) in accordance with the manufacturer’s instructions.