1). Tph1−/− mice displayed no overt physiological differences to WT mice including body weight, food intake, water intake (Supporting Fig. 1), and life span until 30 months of age. The intestines and livers of WT and Tph1−/− mice appeared similar on histology (Supporting Fig. 1). Animals were fed a standard laboratory diet with water and food ad libitum and were kept under constant environmental conditions. All selleck chemicals llc experimental procedures were approved by the Swiss animal welfare authorities and performed in accordance with the institutional animal care guidelines. All animal operations were performed under isoflurane anesthesia. After midline laparotomy, the common
bile duct was freed from the surrounding tissue and dissected between the second and third knot with 8-0 nylon suture for bile duct ligation (BDL). Control animals underwent the same procedure, including the mobilization of the bile duct, but without dissection or ligation. The cystic AZD1152HQPA duct was ligated and
the gallbladder was removed in all animals before BDL to avoid other cholestatic complications (e.g. cholecystitis, bilioma). Thrombocytopenia was induced via intraperitoneal injection of 1 mg/kg anti-CD41 (BD Biosciences). This strategy leads to a reduction of platelets below 10% of their initial count.11 Control mice were treated with an equivalent amount of nonimmune immunoglobulin G (IgG) (BD Biosciences). Blood cell counts were assessed using a Coulter AcT diff counter (Beckman Coulter, Nyon, Switzerland). Serum samples (10 μL) were diluted in distilled deionized water (1:10). A defined amount of glycochenodeoxycholic salt (Sigma) was spiked to the diluted samples and was used as an internal standard. A total of 5 mM ammonium acetate was added to the samples prior to mass spectrometric analyses. Mass spectrometry was performed by direct infusion of the samples as reported.27, 28 Details of the procedure, including the methodology used for the separation of tauro-β-muricholate
by liquid chromatography-mass spectrometry (LC-MS), are described in the Supporting Information. The results are shown as relative amounts versus MCE公司 an internal standard. Isolation of rat hepatocytes was performed following a modified version of the method by Seglen29 and is described in more detail in the Supporting Information. Taurocholate, tauro-β-muricholate (Steraloid Inc.), taurodeoxycholate, taurochenodeoxycholate, and taurolithocholate (Sigma-Aldrich) solutions in Williams’ E medium were mixed at a ratio of 42:18:10:10:20 or 47:40:6:6:1, according to their composition in plasma and liver of cholestatic mice, respectively. Taurolithocholate (TLC) was dissolved with 50% methanol in Williams’ E medium.