Quantifying Intracellular ROS in HUVECs

To detect intracellular ROS, HUVECs stimulated with VEGF were incubated with 10 μM CM–H₂DCFDA [5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester, Invitrogen] for 6 min at 37 °C, fixed with 4% paraformaldehyde for 10 min at room temperature, and then mounted with VECTASHIELD Mounting Medium with DAPI. DCF fluorescence was measured by confocal microscopy (Zeiss) using the identical setting and the same exposure condition in each experiment. Relative DCF fluorescence with DAPI positive cells were analyzed using Image J (NIH). We confirmed that DCF fluorescence was abolished by incubation with adeno-catalase suggesting that DCF signal mainly detects intracellular H₂O₂, as we reported [30 (link)]. In some experiments to detect cellular redox status, we also used cell-permeant fluorescence indicator, CellROX Orange (Invitrogen). Cells were incubated with 5 μM CellROX for 30 min and fluorescence images were taken using confocal microscopy, as previously described with minor modifications [32 (link)].