Zfl cell line

The zebrafish liver (ZFL) cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in a mixed medium (50% L-15, 35% DMEM HG, and 15% Ham’s F12; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) 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), 50 ng/mL epidermal growth factor (Sigma-Aldrich), 5% fetal bovine serum (Gibco), and 0.5% trout serum (Caisson Labs, Smithfield, UT, USA). The cells were incubated at 28 °C in a 100% air atmosphere. The medium was replaced every 2 days.
LD-containing cells were prepared by adding 400 μM lipid mixture (200 μM oleic acid, 100 μM linoleic acid, and 100 μM linolenic acid; coated with bovine serum albumin; Sigma-Aldrich) for 24 h. LD-containing cells were directly incubated with PGF2α and PGE2 (MedChemExpress, Monmouth Junction, NJ, USA; dissolved in dimethyl sulfoxide). Before treatment, cytotoxicity of the molecules was assessed using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay.

The ZFL cell line, obtained from American Type Culture Collection (N° CRL-2634), was derived from normal adult zebrafish [83 (link)]. Cells were grown under humidified air atmosphere at 28 °C in medium composed of 50% Leibovitz L-15 medium, 35% Dulbecco’s modified Eagle’s medium, and 15% HAM’S F-12 medium, supplemented with 5% heat-inactivated FBS; HEPES, 15 mM; NaHCO3, 0.15 g/L; insulin, 0.01 mg/mL; epidermal growth factor, 50 ng/mL, penicillin/streptomycin, 100 U/mL.

The commercially available zebrafish liver (ZFL) cell line (American Type Culture Collection, ATCC, VA, United States) was cultured in culture medium containing 89% DMEM/F-12, 10% FBS, and 1% penicillin/streptomycin at 28°C and 5% CO₂ (i.e., conditions for optimum growth). Lonza Nucleofector (Lonza, Germany) was used for transfection at 70–80% confluence. G418 was added to the transfected ZFL cells after expansion culturing to select for successfully transfected cells. The concentration of G418 for the first week was 1,000 mg⋅mL^–1 and 500 mg⋅mL^–1 thereafter; these were the concentrations at which most of the non-transfected cells died. The G418 was then discarded, and the successfully transfected cells highlighted with green fluorescence.