Black opaque 96 well microplate

HepG2 cells (4×104 cells/well) were incubated in a 6-well plate for 24 h at 37°C. HepG2 cells were then treated with 1 mmol/l FFAs alone or together with compound 2 (25 µmol/l) for 24 h. Subsequently, cells were lysed using an ATP assay kit (cat no. A22026; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions, centrifuged at 12,000 × g for 5 min at 4°C, and the supernatants were collected. Protein concentration was determined using a BCA protein assay kit (Beyotime Institute of Biotechnology) and cells were transferred to a black opaque 96-well microplate (Corning Incorporated). Cellular ATP levels were also assessed using the ATP assay kit (Invitrogen; Thermo Fisher Scientific, Inc.) with the Synergy™ Mx microplate reader (BioTek Instruments, Inc.), according to the manufacturer's protocol (21 (link)).

HepG2 cells (1×104 cells/well) were incubated in a 24-well plate (Costar; Corning Incorporated) for 24 h at 37°C. HepG2 cells at 75% confluence were plated in 24-well plates and were treated with 1 mmol/l FFAs alone or together with 25 µmol/l compound 2 for 24 h. Subsequently, cells were incubated with 10 µmol/l membrane-permeable oxidation-sensitive fluorescent dye DCFH-DA (cat no. D6883; Sigma-Aldrich; Merck KGaA) for 20 min at 37°C. Stained cells were observed under an Eclipse Ti laser scanning confocal microscope (Nikon Corporation, Tokyo, Japan) and photomicrographs were captured. In addition, HepG2 cells were treated with 1 mmol/l FFAs alone or together with compound 2 (25 µmol/l) for 24 h in a black opaque 96-well microplate (Corning Incorporated). Subsequently, cells were incubated with 10 µmol/l DCFH-DA for 20 min at 37°C. During this process, DCFH-DA is cleaved and oxidized to green fluorescent 2′-7-′-dichlorofluorescein via ROS mediation (DCF; excitation/emission, 488/530 nm), the level of which was measured using the Synergy™ Mx microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).