3.10. Intracellular ROS Assay

The intracellular ROS level was measured by fluorescence with a 2′,7′-dichloroflurescein diacetate (DCFH-DA) assay. DCFH-DA without fluorescence can pass through the cell membrane and hydrolyze to generate DCFH. DCFH can be oxidized by intracellular ROS to form fluorescent DCF, while the intensity of green fluorescence is proportional to the level of ROS. PC12 cells were cultured in 6-well plates and pre-treated as mentioned above. Then, the cells were washed with PBS and incubated with DCFH-DA at 37 °C for 20 min. ROS generation was measured by the fluorescence derived from the DCF using a flow cytometer (ACEA Biosciences, San Diego, CA, USA) with excitation and emission wavelengths of 488 and 525 nm, respectively.