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3 protocols using its culture supplement

1

In Vitro Transporter Assay Protocol

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Rifampin, diphenhydramide hydrochloride (98%), dimethyl sulfoxide (DMSO), ammonium acetate, acetonitrile Chromasolv (ACN), formic acid, dexamethasone, diclofenac, thiazolyl blue tetrazolium bromide (MTT), and methanol Chromasolv were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bosentan was purchased from Actelion Pharmaceuticals (Allschwil, Switzerland). Cryopreserved human hepatocytes, HEK293 cells overexpressing OATP1B1, OATP1B3, and OATP2B1 transporter, HEK293 mock cells, phosphate-buffered saline (PBS), Gentest High Viability CryoHepatocyte Recovery kit, hepatocyte culture medium, ITS+ culture supplement, Dulbecco’s Modification Eagle’s Medium (DMEM), Minimal Essential Medium (MEM) non-essential amino acid solution, epidermal growth factor (EGF), and Hank’s balanced salt solution (HBSS) were purchased from Corning (Corning, NY, USA). Penicillin/streptomycin, William’s E medium (without phenol red), fetal bovine serum (FBS), l-glutamine, gentamycin sulfate, SYBR Green ER qPCR Supermix, and Superscript First-strand Synthesis kit were purchased from Life Technologies (Burlington, ON, Canada). Sodium butyrate solution and testosterone were purchased from EMD-Millipore (Darmstadt, Germany) and Acros (Geel, Belgium), respectively. Easy Spin Total RNA extraction kit was purchased from iNtRON (Seongnam, Korea).
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2

Isolation and Culture of Murine Hepatocytes

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Hepatocytes were isolated from 8-week-old C57Bl/6 or C57Bl/6 NOX1−/− mice according to previously described methods [17 (link)]. Briefly, mice were anesthetized with isoflurane and the portal vein was exposed and cannulated with a 26G catheter. The liver was perfused and digested with collagenase type IV (Sigma). Hepatocytes were then purified via Percoll centrifugation and seeded at a density of 1.5 × 105 hepatocytes per well onto 48-well plates coated with 0.17 mg/ml rat tail Collagen-1 (BD Biosciences). Hepatocytes were cultured in media containing DMEM with high glucose (4.5 g/L), 10% (v/v) fetal bovine serum (Biowest), 0.04 µg/ml dexamethasone, 7 ng/ml glucagon, 1% ITS+culture supplement (Corning), 1.5% 1 M HEPES, and 1% penicillin-streptomycin. The next day, hepatocytes were infected. Replicate experiments were performed with both male and female mice.
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3

Cryopreserved Hepatocyte Culture Optimized

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Omeprazole, rifampin, Krebs-Henseleit buffer, mesalazine, and mosapride citrate were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Phenobarbital was purchased from Jeil Pharmaceutical (Daegu, Korea). Cryopreserved human hepatocytes, the Gentest High Viability CryoHepatocyte Recovery Kit, hepatocyte culture medium, epidermal growth factor (EGF), L-glutamine, and matrigel matrix were purchased from BD Bioscience (San Jose, CA, USA). Penicillin/streptomycin, phosphate-buffered saline (PBS, pH 7.4), and gentamicin sulfate were purchased from Gibco® (Burlington, Canada). William’s E medium (No Phenol Red), and GlutaMAXTM were purchased from Invitrogen (Seoul, Korea). The CYP-GloTM CYP 1A2, 2C9, 3A4 assay and the CellTiter-Glo 2.0 assay were purchased from Promega (Madison, WI, USA). ITS+ culture supplement was purchased from Corning (Tewksbury, MA, USA).
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