Insulin transferrin selenium g supplement

A non-tumorigenic mouse hepatocyte cell line AML12 (CRL-2254) was purchased from American Type Culture Collection (Manassas, VA, USA) and maintained in DMEM/Ham F12 (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 1× insulin-transferrin-selenium-G supplement (Invitrogen), 40 ng/mL dexamethasone (Sigma-Aldrich), and 100 ng/mL amphotericin B (Invitrogen). AML12 cells were plated 1 × 10⁴ cells per well in 96-well plates and allowed to adhere overnight. Thereafter, CM or LPS-CM was added, followed by incubation for 24 hours at 37°C. In the TLR4 inhibition test, prior to incubation with CM or LPS-CM, cells were pre-treated with TLR4 inhibitor TAK-242 (1 μM; Calbiochem, La Jolla, CA, USA) or dimethyl sulfoxide (Sigma-Aldrich) as vehicle control for 1 hour. Then, hepatocyte toxicity was induced by maintaining AML12 cells in the presence of 50 mM thioacetamide (TAA) (Sigma-Aldrich). Normal and TAA-treated AML12 cells were incubated with the control medium, LPS (0.5 ng/mL), CM, and LPS-CM, and cell viability was examined by using the Ez-Cytox cell viability assay kit (Daeil Lab Service Co., Ltd, Seoul, Korea).

UMR-106 cells were purchased from ATCC (Manassas, VA) and cultured in high-glucose DMEM containing 10% fetal bovine serum and 1% insulin-transferrin-selenium-G supplement (Invitrogen, Carlsbad, CA).

Human ASCs were kindly donated by Hurim BioCell Co. Ltd. (Seoul, Republic of Korea) (IRB number 700069-201407-BR-002-01). ASCs were plated into culture flask in low-glucose Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, Carlsbad, CA, USA) supplemented with 10% FBS (Thermo Fisher Scientic), 100 U/mL of penicillin (Thermo Fisher Scientic), and 0.1 mg/mL of streptomycin (Thermo Fisher Scientic). The AML12 mouse hepatocyte cell line was obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). AML12 cells were maintained in DMEM/F12 (Dulbecco’s modified Eagle medium/Ham’s F-12; Thermo Fisher Scientic). The medium was supplemented with 10% FBS (fetal bovine serum; GibcoBRL, Calsbad, CA), 1% antibiotics (Thermo Fisher Scientic), 1×ITS supplement (Insulin-Transferrin-Selenium-G supplement; Invitrogen, Calsbad, CA), and 40 ng/mL dexamethasone (Sigma-Aldrich, St. Louis, MO, USA). The LX-2 human stellate cells were kindly donated by Dr. Won-il Jeong in KAIST Biomedical research of Korea. LX-2 cells were maintained in DMEM (Dulbecco’s modified Eagle medium; Thermo Fisher Scientic, Carlsbad, CA). The medium was supplemented with 10% FBS (fetal bovine serum; GibcoBRL, Calsbad, CA), 1% antibiotics (Thermo Fisher Scientic). Cells were incubated at 37 °C in humidified chamber containing 5% carbon dioxide.