5.4. Detection of Reactive Oxygen Species (ROS) or Hydroethidine

Hydroethidine (HE) (Invitrogen) was used to determine HUVECs’ oxidative stress in vitro. Hydroethidine, also called dihydroethidium, exhibits blue fluorescence in the cytosol until being oxidized by ROS to become ethidium and emit a red color. Cells were exposed to HE (2 μM) for 15 min in darkness at room temperature (RT). Then, cells were washed with PBS and fixed with BD CellFIX (BD). Intracellular superoxide levels or intracellular ROS production were quantified and analyzed by flow cytometry.