MTT assay

In order to examine cell proliferation and viability, we performed MTT test. It is a colorimetric assay based on transformation of the yellow MTT tetrazolium salt into purplish formazan crystals due to the metabolic activity of living cells by NADH and NADPH pyridine nucleotide cofactors and mitochondrial dehydrogenases. Thereafter, formazan crystals are dissolved in DMSO and the absorption of the colored solution is quantitatively inspected using an ELISA reader device. In order to perform this experiment, 15 × 10³ cells were seeded on each well of 96-well plates and after 24 h, exposed to various concentrations (40, 80, 120, 160 and 200 μM) of dihydropyrano [2,3-g] chromene solution in DMSO, for 24, 48 and 72 h. Thereafter, 10 μL of MTT (5 mg/mL in PBS) was added to each well. After 4 h, 100 μL of DMSO was used to dissolve the produced formazan crystals. At last, the light absorption of each well was evaluated using an ELISA reader (Awareness technology Inc., USA) at 570 nm (Mosmann 1983).