*4.6. Reactive Oxygen Species Concentration Measurement*

To evaluate the influence of glucose or insulin on reactive oxygen species (ROS) in neuron-like cells, we used an assay with DCF-DA [61], a fluorogenic dye that measures hydroxyl, peroxyl and other ROS activity within the cell. It is deacetylated by esterases to a non-fluorescent compound, which is later oxidized by ROS into highly fluorescent 2 , 7 –dichlorofluorescein. The rest of the supernatant left on the plates used for the Griess assay was removed, and the cell pellet was washed 3 times with PBS. Next, 25 μM of DCF-DA solution in MEM medium, without supplementation or phenol red, was added to the treated PC12 cells and left for 1 h in a CO2-incubator (37 ◦C, 5% CO2, 95% humidity). After incubation, the DCF-DA solution was removed, cells were washed 3 time with MEM, and fresh MEM was added. The fluorescence was immediately measured with excitation at 485 nm and emission at 535 nm using a Varioskan LUX microplate reader (Thermo Scientific). All experiments were performed with 5 wells per concentration and repeated at least 3 times.
