Taurochenodeoxycholate

Standards for primary bile acids cholate (CA) and chenodeoxycholate (CDCA), conjugated primary bile acids glycocholate (GCA), taurocholate (TCA), glycochenodeoxycholate (GCDCA), and taurochenodeoxycholate (TCDCA), secondary bile acids lithocholate (LCA), deoxycholate (DCA), ursodeoxycholate (UDCA), and hyodeoxycholate (HDCA), and conjugated secondary bile acids glycolithocholate (GLCA), taurolithocholate (TLCA), glycodeoxycholate (GDCA), and taurodeoxycholate (TDCA) were purchased from Sigma.

Bovine bile (reference B3883, Sigma-Aldrich and reference 212820, Difco, BD Diagnostic Systems) was prepared at 100 mg/ml stock solution in distilled water, sterilized through 0.2 μm filters and stored at −20°C. Pure bile salts: glycocholate (GC), taurocholate (TC), glycodeoxycholate (GDC), taurodeoxycholate (TDC), glycochenodeoxycholate (GCDC), and taurochenodeoxycholate (TCDC), bile salt mix or pure deconjugated bile salts: cholate (C), deoxycholate (DC), and chenodeoxycholate (CDC), and fusidic acid were from Sigma-Aldrich and dissolved in distilled water to 12 mg/ml stock solutions, filtered through 0.2 μm pores and kept at −20°C. Choloylglycine Bile Acid Hydrolase or Bile Salt Hydrolase (BSH, EC 3.5.1.24) from Clostridium perfringens (reference C4018, Sigma-Aldrich) was prepared at 10 U/ml in distilled water and stored at −20°C. Fetal calf serum (FCS, reference A15-101, PAA Laboratories, GE Healthcare), and bovine calf serum (reference B9433, Sigma-Aldrich) were used. The NEFA-C kit used for quantitative determination of non-esterified fatty acids (NEFAs) was from Biolab, WAKO Diagnostics.

HepG2 cells were obtained from ATCC. Cells were maintained in DMEM supplemented with 10% fetal calf serum (FCS), penicillin and streptomycin in. To obtain BMDCs, bone marrow was isolated from C57Bl/6 mice. Approximately 2 × 10⁶ cells were seeded on culture dishes with 10% FCS in RPMI medium with GM-CSF (40 ng/ml) for 10 days. Cells were harvested on day 10 and treated with GRP78 (BioMatik) and poly I:C (50 μg/ml) (Sigma). Apoptosis was assessed using Annexin V and 7AAD staining (Thermofisher). HepG2 cells were treated with glycocholic Acid (GCA), glycochenodeoxycholic acid (GCDC), taurocholic acid (TCA) and taurochenodeoxycholate (TCDC) (All from Sigma) indicated concentrations.