MTT assay for cell viability

Cell viability was measured using the MTT assay as previously described [27], and based on the 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, M2003; Sigma, St. Louis, MO, USA) conversion into formazan crystals using mitochondrial dehydrogenases. The prepared H9c2 cells suspension was seeded into 96-well plates at a density of 1 × 10⁴ cells/well and incubated at 37 °C for 24 h. Then the cells were treated with (0-300 μg/ml) AGEs as indicated for (0-36 h). The supernatant was discarded and the culture medium was replaced with 100 μl of MTT solution (5 mg/ml stock solution in DPBS, diluted with culture medium to the final concentration 0.5 mg/ml) was added into each well. Then the cells were maintained at 37 °C for 4 h, this solution was removed and the formed formazan was dissolved with 100μl dimethyl sulfoxide (DMSO). The optical density (O.D.) values of samples were read at 580 nm with a micro plate reader.