2.2.1. Neuro-2a Cell Culture and Treatments with Urolithin A and Hydrogen Peroxide

Neuro-2a cells (ATCC® CCL-131™) were thawed and cultured in 10% FBS-supplemented DMEM and 1% penicillin-streptomycin, seeded in a T175 flask, and placed into the incubator (5% CO2, 37 ◦C). Once they reached the state of confluence, they were sub-cultured in a 96-well plate at a density of 1 × 10<sup>4</sup> cells/well in DMEM (10%) and incubated (37 ◦C, 5% CO2) for 24 h before the treatment.

Stock solutions of urolithin A (438 mM) were prepared in sterilized PBS containing 1% DMSO (final concentration in the cells less than 0.1%). The sample was vortexed and filtered with a 0.22-μm syringe filter. Five dilution series were done from the stock solution. Stock solutions of hydrogen peroxide (1000 μM) were prepared and incorporated into the cells at a final concentration of 250 μM (45 min) to induce oxidative stress in the cells.
