*4.5. Determination of Reactive Oxygen Species (ROS) and Oxidative Stress Status*

The intracellular ROS amounts were determined using a fluorometric assay with 2,7-dichlorofluorescein diacetate (DCFHDA). After diffusion inside the cell, the probe is hydrolyzed by intracellular esterase to non-fluorescent dichlorofluorescein (DCFH) and then oxidized to fluorescent DCF in the presence of ROS [66]. PC12 cells were seeded on 24-well culture plates at 105 cells/well for 24 h. After incubation, cells were treated with 20 μM DCFHDA. Intracellular production of ROS was measured after 30 min incubation at 37 ◦C by fluorometric detection of DCF oxidation on a fluorimeter with an excitation wavelength of 485 nm and emission wavelength of 522 nm. Results are expressed as the ratio of intensity of fluorescence in treated cells to that of the HE responding fluorescence in the control.
