Intracellular ROS Generation Assay

To assess the intracellular generation of reactive oxygen species (ROS), cells were plated in 96-well plates and allowed to adhere overnight. Cells were treated with MG and incubation with 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorochrome (Molecular Probe; Thermo Fisher Scientific, Inc., Life Technologies Italia, Monza, Italy) was performed as previously reported [37 (link)]. For this purpose, stock solutions of H2DCFDA were dissolved in DMSO and aliquots were stored at −20 °C until use. H2DCFDA working solution was prepared in a PBS solution containing 5 mM glucose and added to cells to the final concentration of 20 μM. Then, incubation was protracted for 30 min in the dark and ROS-positive cells were visualized using a fluorescein isothiocyanate (FITC) filter (excitation wavelength of 485 nm and emission wavelength of 530 nm) in a Leica inverted fluorescence microscope (Leica Microsystems S.r.l, Wetzlar, Germany) equipped with a DC300F camera. All pictures were captured using Leica Q Fluoro software (Leica Microsystems S.r.l, Wetzlar, Germany).