2.6. Intracellular ROS measurement

Intracellular ROS was measured by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) assay. DCFH-DA assay is based on the ROS-dependent oxidation of 2′,7′-dichlorodihydrofluorescein (DCFH) to the highly fluorescent compound 2′,7′-dichlorofluorescein (DCF). In brief, BV-2 microglia cells in 96-well plates were rinsed in PBS, incubated with DCFH-DA (10 μM) for 30 min at 37 °C in the dark. Fluorescence was measured in a fluorescence microplate reader (Spectra Max Gemini EM, Molecular Devices, Sunnyvale, CA, USA) at excitation wavelength 485 nm and emission wavelength 535 nm. The cells were also pictured by using laser-scanning confocal microscope (FV1000, Olympus, Japan) equipped with FV10-ASW 4.0 VIEW (Olympus), and the investigators were blinded to the treatment.