2.11. Superoxide measurements

Dye dihydroethidium (DHE; Molecular Probes, Eugene, OR, USA) was applied for oxidative fluorescent, which was detected with confocal microscopy as described previously [22]. In brief, unfixed frozen aortic segments were stained with 10-6 mol/L DHE at 37 °C for 1 h in a dark humidified container. Then, DHE fluorescence was detected using a confocal microscope (Olympus, Tokyo, Japan) via a 590 nm long-pass filter.

Intracellular superoxide levels were detected in HUVECs with 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), as described previously. Briefly, After 30 min incubation with CM-H2DCFDA (20 μM) at 37 °C, cells fluorescence was detected by a Synergy HT microplate reader (BIO-TEK) with excitation set at 490 nm and emission detected at 520 nm.