Quantichrom bilirubin assay kit

The concentration of BR in whole cell lysates were measured by QuantiChrom™ Bilirubin Assay Kit (DIBR-180, BioAssay Systems, California, USA) according to the manufacturer's protocol. The color change was detected at 530 nm wavelength. The BR level was calculated as [OD_sample − OD_blank]/[OD_calibration − OD_H2O] × 5 mg/dL.

Gene expression of uridine 5′-diphospho-glucuronosyltransferase 1A1 (UGT1A1), membrane metalloendopeptidase (MME), ATP-binding cassette transporter B1 (ABCB1), ABC11, and ABCC2 was analyzed in SHED-Heps by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), as described in the Additional file 1: Supplementary Methods. Distribution of MME, ABCB1, ABCB11, and ABCC2 in SHED-Hep spheroids was analyzed by immunohistochemistry. To analyze hepatobiliary function assays, SHED-Heps were incubated with indirect bilirubin (25 μM; Merck, Darmstadt, Germany) in Ca²⁺-free HBSS (Nacalai Tesque) at 37 °C for 60 min and used for determining direct bilirubin by colorimetric assay using QuantiChrom Bilirubin Assay Kit (BioAssay Systems) according to the manufacturer’s instructions. SHED-Heps were also incubated with CLF (5 μM; Corning) in Ca²⁺-free Hank’s balanced salt solution (HBSS; Nacalai Tesque) at 37 °C for 15 min. Intact SHED and human intrahepatic biliary epithelial cells (AXOL, Cambridge, UK) were used as controls.

GSH, GSSG, Sepasol-RNA I Super G, and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Nacalai Tesque Inc. (Kyoto, Japan). ReverTra Ace was procured from Toyobo Co., Ltd. (Osaka, Japan). Fast SYBR Green Master Mix and the BCA protein assay kit were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Transaminase CII-test Wako and MS-grade porcine pancreatic trypsin were obtained from Fujifilm Wako Pure Chemical Co., Ltd. (Osaka, Japan). The QuantiChrom bilirubin assay kit was obtained from BioAssay Systems, LLC. (Hayward, CA, USA). Percoll was obtained from Cytiva (Tokyo, Japan). LPS from Escherichia coli was procured from Sigma-Aldrich Co., LLC. (St. Louis, MO, USA). LPS-RS was obtained from InvivoGen Inc. (San Diego, CA, USA). FPS-ZM1 was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Oligonucleotide primers were obtained from Eurofins Genomics Inc. (Luxembourg, Luxembourg). All other chemicals and solvents were of MS grade or higher and of commercially available purity.