ZFL cells were cultured as previously reported [21 (link)]. Briefly, cells were cultured in a mixed medium containing 50% L-15, 35% DMEM HG, and 15% Ham’s F12 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), which was supplemented with 0.15 g/L sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA), 15 mM HEPES (Sigma-Aldrich), 0.01 mg/mL bovine insulin (Sigma-Aldrich), 50ng/mL epidermal growth factor (Sigma-Aldrich), 5% fetal bovine serum (Gibco), and 0.5% trout serum (Caisson Labs, Smithfield, UT, USA). The cells were cultured in a cell incubator set at 28 °C.
For the LD formation experiment, the cells were incubated either with NM or with high-fat medium (HFM) containing 300 μM bovine serum albumin (BSA)-coated oleic acid (Sigma-Aldrich) for 6 h. The BSA-coated oleic acid is obtained by adding the oleic acid dissolved in ethanol dropwise to the BSA solution to obtain a homogenous mixture. For the retinyl acetate treatment experiment, 10 μM retinyl acetate (MedChemExpress, Monmouth Junction, NJ, USA) was added to cells cultured in NM or HFM, followed by incubation for 6 h.
For the LD formation experiment, the cells were incubated either with NM or with high-fat medium (HFM) containing 300 μM bovine serum albumin (BSA)-coated oleic acid (Sigma-Aldrich) for 6 h. The BSA-coated oleic acid is obtained by adding the oleic acid dissolved in ethanol dropwise to the BSA solution to obtain a homogenous mixture. For the retinyl acetate treatment experiment, 10 μM retinyl acetate (MedChemExpress, Monmouth Junction, NJ, USA) was added to cells cultured in NM or HFM, followed by incubation for 6 h.