Cholyl lysyl fluorescein clf

To assess uptake of low-density lipoprotein (LDL), SHED-Heps were incubated with acetylated LDL labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindo-carbocyanine perchlorate (DiI-Ac-LDL; 10 μg/mL; Biomedical Technologies, Madrid, Spain) for 4 h at 37 °C. To examine transport of bile acid, SHED-Heps were incubated with cholyl-lysyl-fluorescein (CLF; 5 μM; BD Bioscience, Franklin Lake, NJ) in HBSS at 37 °C for 15 min. SHED and HepG2 cells (Riken) were used as the negative and positive controls, respectively.

Male C57Bl6/N 6–8 week-old (Janvier Labs, France) or mT/mG reporter (Muzumdar et al. 2007 (link); The Jackson Laboratory, ME, USA) mice were used in this study. The mice were housed under 12 h light/dark cycles at controlled ambient temperature of 25 °C, and were fed ad libitum with standard diet (Ssniff, Soest, Germany) and had free access to water. The experiments were approved by the local authorities. Aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) were purchased from Sigma-Aldrich, Darmstadt, Germany (Cat. No. A6636 and O1877, respectively). OTA was also isolated from fungal cultures as described by Bittner et al. (2013 (link)) and provided by the Institute of Food Chemistry, WWU Münster, Germany. Tetramethylrhodamine–ethyl ester (TMRE) was purchased from Thermo Scientific, MA, USA (Cat. No. T669). Cholyl–lysyl–fluorescein (CLF; a bile acid analog) was obtained from BD Biosciences, California, USA (Cat. No. 451041).

BA-SHED-Heps and Cont-SHED-Heps were incubated with indocyanine green (ICG; 1 mg/mL; Merck) in IMDM (Thermo Fisher Scientific) for 1 h. After washing with HBSS (Nacalai Tesque), the cells were cultured for 6 h. The cells were incubated overnight with 0.1% bovine serum albumin (BSA; Merck) in IMDM (Thermo Fisher Scientific) and treated for 5 h with Dil-conjugated acetylated low-density lipoprotein (Dil-Ac-LDL; 10 μg/mL; Cell Applications, San Diego, CA, USA) and 0.1% BSA (Merck) in IMDM (Thermo Fisher Scientific). BA-SHED-Heps and Cont-SHED-Heps were also incubated with cholyl-lysyl-fluorescein (CLF; 5 μM; BD Bioscience, Franklin Lake, NJ, USA) in HBSS (Nacalai Tesque) at 37 °C for 15 min. All samples were fixed with 4% paraformaldehyde and stained with DAPI (1 µg/ml; Thermo Fisher Scientific). All samples were imaged with an Axio Imager M.2 fluorescent microscope equipped with an Apotome 2 (Carl Zeiss Microscopy). Cont-SHED and BA-SHED were used as controls.