Measurement of Intracellular ROS

ROS are produced at the beginning of the inflammation phase. Therefore, the effect of CAT on intracellular ROS accumulation was measured. The generation of intracellular ROS was determined using DCFH-DA fluorescent dye (Wang and Joseph, 1999). The DCFH-DA probe is a non-polar compound which lightly diffuses into cells, where it is hydrolyzed by intracellular esterase to generate DCFH which is trapped within the cells and becomes intracellularly oxidized to form the highly fluorescent compound 2, 7-dichlorofluorescein (DCF) which is measured flow cytometrically. In this assay, primary cultured cortical neurons were seeded in six-well plates. Following various treatments, the cells were incubated with 10 μM DCFH-DA for 30 min at 37°C in the dark. Subsequently, the cells were harvested, washed twice with PBS, and re-suspended for fluorescence analysis using a flow cytometer.