Humidified tissue culture incubator

HepG2 and BRL3A cell lines were all from American Type Culture Collection (ATCC, Manassas, VA, USA). Human hepatocellular carcinoma cell HepG2 and rat liver fibroblast BRL3A were cultured in DMEM added with 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL). All cell lines were grown in humidified tissue culture incubator (SANYO) with an atmosphere of 5% CO₂ at 37 °C. The method concerning a high-fat medium reported by Lee et al. [36 (link)] was preferred. The free fatty acid (FFA) mixture (oleic acid/palmitic acid, 1:1) was prepared with fat-free bovine serum albumin (BSA, B2064, sigma, St. Louis, MO, USA). After attaining 70% confluence, cells were cultured with normal medium or exposed to medium containing 1 mmol/L FFA, with or without 10 mmol/L betaine, for 24 h. The level of TC in cells was measured by a cholesterol cell-based detection assay kit (No. 10009779, Cayman, Ann Arbor, MI, USA). The level of cholesterol uptake was determined by a cholesterol uptake cell-based assay kit (No. 600440, Cayman, Ann Arbor, MI, USA) according to the operation manual.

Mouse kidney stem cells were purchased from AcceGen (ABC-SC0037, Fairfield, CT, USA) and cultured in 3 mL of a MOUSE KIDNEY STEM CELL Growth Medium Kit (ABM-SM0037, AcceGen, NJ, USA) in a T-25 culture flask (70025, SPL, Pocheon, Republic of Korea). The cells were incubated in a 5% CO2 and 37 °C humidified tissue culture incubator (Sanyo, Osaka, Japan). The human embryonic kidney cells (HEK293F) were purchased from Invitrogen (Waltham, MA, USA) and were grown in DMEM (LM001-05, WELGENE, Gyeongsan, Republic of Korea) containing 10% FBS, 50 μg/mL plasmocin (InvivoGen, San Diego, CA, USA), 100 unit/mL penicillin, and 100 mg/mL streptomycin (L0022, Biowest, Nuaillé, Frances).