2.2.2. Mitochondrial Activity in Neuro-2a Cells Subjected to Oxidative Stress after Urolithin A Treatment

Cells were cultured in 96-well plates at a concentration of 1 <sup>×</sup> <sup>10</sup><sup>4</sup> cells per well. The cytotoxicity of urolithin A (0.5 μM–20 μM) was measured after 24 h by adding MTT (3-(4,5-dimethylthiazol -2-yl)-2,5-diphenyltetrazolium bromide) to the cell culture. Additionally, this assay can be performed to assess the potential cytoprotective effect of urolithin A after inducing a neuronal injury with 250 μM hydrogen peroxide for 45 min. After treatments (24 h exposition to urolithin, 45 min to hydrogen peroxide), cells were incubated for an additional 24 h; DMEM was then removed from every well and replaced with MTT solution (2 mg/mL in DMEM) and incubated at 37 ◦C for 3 h. Finally, MTT solution was removed and 100 μL of DMSO was added in each well to dissolve formazan crystals. Via a Synergy H1 Hybrid Multi-Mode Reader (Biotek, Bad Friedrichshall, Germany), the absorbance was read at 550 nm. Experiments were carried out in different weeks and diverse passages and expressed as percentage of control.
