2.4. Determination of ROS

It is known that dihydroethidium (DHEt) diffuses into cells and is oxidized by reactive oxygen species in the cytosol producing ethidium and 2-hydroxyethidium that binds to the DNA and emits bright red fluorescence. Thus, fluorescence intensity is an indirect parameter of ROS levels. CGN were maintained in a K25 medium during 7 days in vitro (DIV) and after this time the CGN were transferred to a medium with K5 or treated with Sts and maintained in these conditions from 0.5 h to 5.5 h. After treatment, the cells were incubated with 3.2 µM DHEt for 20 minutes at 37°C. Then, cells were washed with PBS and observed in a fluorescence microscope with a rhodamine filter with a wavelength of 488–515 nm. Cells were photographed and fluorescence intensity was measured with the Image J Program. ROS production by xanthine/xanthine oxidase was used as positive control. For that, 7-8 DIV cultures were incubated with 10 µM xanthine (X) for 1 h and then xanthine oxidase (XO) (45 mU/mL) was added to the medium and ROS generation was evaluated after 2 h [35].