2.7.2. MitoSOX Fluorescent Assay

The effect of the plant extracts on mitochondrial superoxide anion was determined using the fluorescence dye[49] MitoSOX Red, a cationic derivative of dihydroethidium (DHE), which reacts with superoxide anion in the mitochondrial matrix [51]. MitoSOX Red is oxidized by mitochondrial superoxide anion to form 2-hydroxymitoethidium, which excites and emits at 510 and 580 nm, respectively, and exhibits red fluorescence [33]. This assay's cell handling procedure was similar to the ones used for the SRB and the resazurin assays (Section 2.6.1). After pretreatment with the extracts and treatment with Dox, the medium was removed, and the cells were washed twice with PBS. Then, 50 µL of MitoSOX working solution (5 µM in basal medium) was added to the cells, followed by an incubation with the dye solution for 20 minutes at 37°C. The fluorescence was read for 90 min, at 37°C, with 510 nm excitation wavelength and 580 nm emission wavelength using a Cytation 3 multiplate reader. The cells were then fixed with ice-cold methanol in acetic acid, and the SRB assay was performed to normalize the results.