Measurement of cellular ROS

Intracellular ROS levels (superoxide anion, ·O₂₋) were measured by using dihydroethidium (DHE), which can be oxidized by ·O₂₋ into 2-hydroxyethidium and exhibits red fluorescence (Ex/Em=518/605 nm; Invitrogen). Differentiated N2a cells (1.5 × 10⁴ in each well of a 96-well plate) were treated with 50 μM H₂O₂ (1h up to 3h). Then, the cells were washed once with pre-warmed PBS supplemented with 10 μM DHE. After incubation at 37 °C for 30 min, cells were washed again and DHE fluorescence was detected by the FL2 laser of a flow cytometer (BD FACSCalibur), and analyzed by BD CellQuest software.