Quantifying Cellular Oxidative and Nitric Oxide Levels

Chloromethyl-dichloro-dihydro-fluorescein diacetate (DCFDA, Molecular Probes, Life Technologies, USA) was used for ROS detection, and 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM, Molecular Probes, Life Technologies, USA) for NO detection as fluorescent probes, as described in our previous studies [39 (link), 40 (link)]. Brain endothelial cells were cultured in 96-well plates with black walls and transparent plastic bottoms (Corning, NY, USA). After treatments for 1 or 24 h, cells were incubated in Ringer–Hepes buffer containing 2 µM DCFDA or 2 µM DAF-FM probes. Pluronic acid (Molecular Probes, Life Technologies, USA; 16 µM) was used to help the probes crossing the cell membrane. Fluorescence was detected by Fluostar Optima multiwell plate reader (BMG Labtechnologies, Germany) at 485 nm excitation and 538 nm emission wavelengths at every 3 min for 1 h.