“Redox Imaging” to Distinguish Cells with Different Proliferative Indexes: Superoxide, Hydroperoxides, and Their Ratio as Potential Biomarkers

Figure 6

(a) Biochemical strategy to enhance superoxide accumulation in cells by inhibition of mitochondrial electron transport chain and mitochondrial superoxide dismutase (SOD2) (according to Pelicano et al. [44]). (b) Level of superoxide and activities of antioxidant enzymes in cells, treated with 2-ME/Rot. Experimental conditions: cells (normal lymphocytes; cells/mL) were preincubated in the absence or presence of 600 nM 2-ME and 500 nM Rot within 6 hours in a humidified atmosphere (5% CO₂, 37°C). Control sample contained untreated cells. The superoxide level was measured by DHE assay, and the activities of antioxidant enzymes were measured as described in Materials and Methods. (c) EPR signal intensity of mito-TEMPO in untreated and 2-ME/Rot-treated cells. Experimental conditions: cells ( cells/mL) were preincubated in the absence or presence of 600 nM 2-ME and 500 nM Rot within 6 hours in a humidified atmosphere (5% CO₂, 37°C). Mito-TEMPO (0.1 mM) was added to the cell suspensions, and the incubation was continued for 1 hour at the same conditions. Aliquots of the cell suspensions were collected and subjected to EPR analysis. Control sample contained mito-TEMPO (0.1 mM) in cultured (cell-free) medium. In (b) and (c), the data are the from three and four independent experiments, respectively. and versus control; versus cells only. Dotted red lines show the control levels. ME: 2-methoxyestradiol; Rot: rotenone; SOD: superoxide dismutase; GSH-Px: glutathione peroxidase.

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