4.6. Measurement of Intracellular Free Radicals Level

Measurement of the free radicals level was carried out using fluorescent indicator 2'7'-dichlorofluorescein diacetate (DCFH-DA), as described previously [30]. DCFH-DA is intracellularly deacetylated to 2'7'-dichlorofluorescin (DCFH) and then oxidized by hydrogen peroxide to a fluorescent compound, 2'7'-dichlorofluorescein (DCF). SH-SY5Y cells were incubated in DCFH-DA (10 µM) solution in HBSS with 20 mM HEPES (pH 7.4) and 0.02% Pluronic for 50 min at 37 °C in the dark. Then the cells were washed three times, and the DCF fluorescence was measured using a microplate reader FLUOstar Omega (Ortenberg, Germany) at 485 nm excitation and 538 nm emission wavelengths. After determining the baseline fluorescence of the cells incubated in HBSS, the changes in fluorescence after the addition of the test compounds were recorded every 1 h for 8 h. The results of fluorescence measurements are presented as percent of corresponding control.