*4.5. Determination of ROS*

The intracellular level of ROS, as an important biomarker for oxidative stress, was estimated with the dye 2 7 -dichlorodihydrofluorescein diacetate (H2DCFDA; D399, Molecular Probes, Invitrogen, Paisley, Scotland, UK), a nonpolar compound that easily penetrates into the cell where it is hydrolyzed to the nonpermeant H2DCF. This nonfluorescent compound becomes oxidized by various ROS to highly fluorescent 2 ,7 -dichlorofluorescein (DCF). For determination, 3000–5000 cells/well were seeded in 96-well plates and grown in 100 μL/well of complete medium. Once treatments were completed, medium was removed and prewarmed PBS containing the probe (final working concentration of 10 μM dye) was added to the cells. After incubation for 60 min at 37 ◦C, the dye was removed and cells were returned to prewarmed growth medium. Then, fluorescence was measured in a microplate fluorimeter FLX-800 (Bio-Tek Instruments, Inc., Winooski, VT, USA) at an excitation wavelength of 485 nm and an emission wavelength of 528 nm.
