Mouse total bile acids assay kit

Bile acid profiling was performed as described.^{3 (link),12 (link)} Livers were homogenized in water (100 mg tissue in 500 μL water), and then, 300 μL of methanol:acetonitrile (v/v, 1:1) was added to a 100 μL aliquot of liver homogenate. All the mixtures were vortexed for 2 minutes and centrifuged at 15,000 rpm for 10 minutes. Two microliters of the supernatants from all samples were injected into the ultraperformance liquid chromatography coupled with an SYNAPT G2-S quadrupole time-of-flight mass spectrometry (QTOFMS, Waters Corporation, Milford, MA). The column type is Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm). Protocols for the use of a mobile phase gradient and QTOFMS system were reported.^{12 (link),13 (link)} Bile acid species were quantified by measuring their relative abundance as the AUC for each species using standards for comparison.
Serum bile acid quantification was done using the Mouse Total Bile Acids Assay Kit (CrystalChem #80471). Serum samples were diluted 1:5 and were analyzed per kit instructions.

Triglyceride content was determined on ~25 mg of flash-frozen tissue using the Infinity™ Triglycerides reagent (Thermo Scientific, USA) as previously described [50 (link)]. All assays were performed on at least 6 sets of tissue.
To determine serum and hepatic sterol levels, we used ion-ratio GC/MS on an Agilent 6390N/5973 GC/MS system on 7 sets of mice as previously described [51 (link)] with modifications to the GC/MS method to include ions specific for principal post-squalene cholesterol precursors. Bile acids were quantified on frozen total liver tissue using a Mouse Total Bile Acids Assay kit (Crystal Chem, Downer's Grove, IL) using the suppliers' protocol.
All statistical analysis was implemented using a simple Students' t-test unless otherwise noted.

Feces were weighed out. Bile acids were extracted using 90% ethanol with 0.1N NaOH. Bile acids were determined using Mouse Total Bile Acids Assay Kit (Crystalchem, catalog 80471) according to the manufacturer’s instructions.

Total bile acids from plasma and gallbladder bile were measured with Mouse Total Bile Acids Assay Kit (Crystal Chem, Zaandam, Netherlands) according to the manufacturer’s protocol.
Individual bile acids were measured from plasma. To remove plasma proteins, 50 µl of EDTA-plasma was mixed with 500 µl of acetonitrile, kept for 1 h at −20 °C and centrifuged. The supernatant was dried in a vacuum and redissolved into 50 µl of 50% acetonitrile. 5 µl of the solution was analyzed by LC/MS using Aquity UPLC system (Waters, Milford, MA, USA) connected to Synapt G1 Q-TOF mass spectrometer (Waters). The eluents were A: 5 mM ammoniumacetate, 0.018% formic acid and B: 5% acetonitrile in methanol. The linear gradient was operated at 0.4 ml/min with the following program: 0 min 40% B; 10 min 100% B, 14 min 100% B, 15 min 40% B. The column, BEH C18, 1.7 µm, 2 × 100 mM (Waters), was kept at 40 °C. The mass spectrometer collected 0,2 second scans in the mass range between 50–1500 in V-mode using negative ionization, recording centroid peaks with lock mass (Leu-encephalin) correction. Quantification was calibrated with standards at 1, 5, 10, 30, 70 and 100 ng/ml. Standards were cholic acid (C1129; Sigma), chenodeoxycholic acid (C9377, Sigma), deoxycholic acid (D2510, Sigma) and tauro α-muricholic acid (C1893–000, Steraloids Inc., Newport, RI, USA).