Cflow software v 264

ROS are oxygenic free radicals which can alter the balance of endogenous protective systems, such as glutathione and enzymatic antioxidant defense systems (17 (link)). ROS production in cells was detected by a standard, cell permeable, fluorescent dye method, 2,7-dichlorofluorescin diacetate (DCFH-DA). Intracellular esterases hydrolyze DCFH-DA to 2,7-dichlorofluorescin (DCFH), which is then oxidized by ROS to dichlorofluorescein (DCF), and therefore amount of fluorescence reflects ROS production in cells. Fluorescence intensity was measured by Accuri™ C6 flow cytometry (BD Biosciences) with BD CFlow Software v.264.15 (BD Biosciences) at excitation and emission wavelengths of 485 nm and 530 nm, respectively (18 (link)).

Following treatment with 0, 20 or 40 µM POA for 24 h, 5×10⁵ cells were collected by centrifugation at 800 × g for 5 min at 4°C, washed twice with PBS, and then fixed with 70% chilled ethanol for 12 h. Following fixation, cells were washed twice with PBS and incubated in PBS containing 50 mg/ml PI, 1 mg/ml RNase A and Triton X-100 (0.5%) at 4°C for 30 min in the dark. The fluorescence emitted from the PI-DNA complex was measured using Accuri™ C6 FACScan flow cytometry (BD Biosciences) with BD CFlow Software v.264.15 (BD Biosciences). The cells with nuclei with sub-G1 content were considered apoptotic cells (9 (link)).

ROS accumulation in cells was measured by a ROS detection kit (Nanjing Jiancheng Bio Institute) using the cell permeable fluorescent dye 2,7-dichlorofluorescin diacetate (DCFH-DA). The intracellular esterases hydrolyze DCFH-DA to DCFH, which is oxidized to DCF by the oxidants; its fluorescence is a measure of ROS production in the cell. A BD flow cytometer with BD CFlow software v.264.15 (BD Biosciences) was used to measure the fluorescence intensity at excitation and emission wavelengths of 485 and 530 nm, respectively.