2.8. Dihydroethidium (DHE) measurement

DHE was evaluated as a surrogate of reactive oxygen species (ROS) production in live BCAEC. DHE was used as a fluorescent probe for the detection of superoxide and hydrogen peroxide. Antimycin A, an inhibitor of complex III of the mitochondrial electron transport chain, was included as a positive control for ROS generation. N-acetyl Cysteine was included as an anti-oxidant control. Breifly, cells were seeded in a 96 well plate at a density of 50 cells/well. After 48 h treatment with compounds cells were washed with PBS and added 150 μl of Cell-Based Assay Buffer and incubated for 10 min at room temp. Cell-Based Assay Buffer, was removed and 20 μl of fresh assay buffer and 130 μl of ROS Staining Buffer per well were added. Then 10 μl of N-acetyl cysteine assay reagent were added to designated negative control wells and incubated for 30 min at 37 °C protected from light. Following, we added 10 μl of the Antimycin A reagent to positive control wells and incubated for 1 h at 37 °C protected from light. Carefully we aspirated ROS Staining Buffer and added 100 μl of Cell-Based Assay Buffer. Plate was placed on fluorometer (FLx800, Bio-Tek Instruments) plate reader and fluorescence was measured at an excitation wavelength between 480 and 520 nm and an emission wavelength between 570 and 600 nm. ROS generation data was represented as total DHE fluorescence units.