Measurement of ROS production
To measure the oxidative stress produced in the astrocytes, the cells were treated with various agents for appropriate duration. After the termination of treatments, the cells were washed two times with PBS and incubated with 5 μM 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) (Molecular Probes, Carlsbad, CA, USA) for 30 min in serum-free medium at 37 °C. The cells were then washed two times with PBS to remove unloaded dye and the images were taken using Leica fluorescent microscope (DMI 3000B; Leica Microsystems Inc., Buffalo Grove, IL, USA). Unstained cells were used as experimental controls and the fluorescent images were obtained using excitation and emission wavelengths at 485 and 535 nm, respectively.
To obtain the fluorescence intensity corresponding to ROS, the cells were collected and acquired using FITC wavelengths on FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA). The ROS production was measured using unstained cells as negative controls and cells treated with 500 μM H2O2 as positive controls. The mean fluorescence intensities were compared between different treatments.