The cells were seeded in 96-well culture plates at a density of 1 × 104 cells/well for 24 h. The culture medium was changed to fresh MEM containing 2% FBS and 1% antibiotics, and for hypoxic preconditioning COCl2 was added to the complete medium at a concentration of 100 μM; COCl2 was purchased from Sigma (Gabriella et al. 2018 ).
After 24 h, the medium was changed with a new one supplemented with 0.5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 3 h at 37 °C. The formazan produced was dissolved by solvent solution (0.1 N HCl in isopropanol), and the optical density was read at 570 nm by a microplate reader (Model 680, Bio-Rad Lab Inc., CA, USA) (Lan et al. 2015 (link)).
After 24 h, the medium was changed with a new one supplemented with 0.5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 3 h at 37 °C. The formazan produced was dissolved by solvent solution (0.1 N HCl in isopropanol), and the optical density was read at 570 nm by a microplate reader (Model 680, Bio-Rad Lab Inc., CA, USA) (Lan et al. 2015 (link)).
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