Dmem ham s f12 medium

GT1-7 cells are immortalized hypothalamic neurons derived from mice and were provided by Dr. R. Weiner (University of California, San Francisco, CA, USA). GT1-7 cells were cultured in DMEM/Ham’s-F12 medium supplemented with 10% fetal bovine serum (South American origin) (Biowest, Nuaillé, France). After treatment with trypsin (Fujifilm Wako Pure Chemicals), the cells were suspended in a serum-free medium, seeded into culture plates, and cultured in a humidified incubator (7% CO₂) at 37 °C [19 (link)].

The mouse chondrogenic cell line ATDC-5 was kindly gifted by Dr. Oreste Gualillo (University of Santiago de Compostela, La Coruña, Spain). Cells were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM)–Ham’s F-12 medium (Biowest, Nuaillé, France) supplemented with 5% FBS (Gibco, UK), 10 μg/mL human transferrin (Sigma-Aldrich, St. Louis, USA), 3 × 10⁻⁸ M sodium selenite (Sigma-Aldrich, St. Louis, MO, USA), 1% penicillin-streptomycin-amphotericin B (Biowest, Nuaillé, France), and 1% stable glutamine (Biowest, Nuaillé, France) in a humidified atmosphere of 5% CO₂ at 37 °C.
The viability of cells was assessed using the Blue Cell Viability Assay (Abnova, Taipei, Taiwan). In brief, cells (10⁴ cells/well) were cultured in 96-well plates and treated with onion extracts (OE) (1.9–62.5 µg/mL) for 24 h at 37 °C. After that, the reagent was added in accordance with the manufacturer’s instructions (10%; incubation of 4 h at 37 °C and 5% CO₂). Metabolically active cells were able to reduce the dye, and the fluorescence generated was calculated in a microplate reader (Varioskan Lux, Thermo Fisher, Waltham, MA, USA) at 530 nm excitation and 590 nm emission wavelengths. Viability percentages were determined by linear interpolation of data considering control samples (non-treated cells) as 100% of viability.