2.6 Measurement of intracellular reactive oxygen specie levels in SH-SY5Y cells

Briefly, following the manufacturer’s instructions, using 2,7-dichlorodihydrofluorescein diacetate and dimethyl sulfoxide as stock solutions, intracellular ROS levels were measured as described previously (Zhou et al., 2017). Before treatment, SH-SY5Y cells were seeded in 6-well plates for at least 12 h. Incubation with or without reserpine (100 μmol/L) was for 24 h followed by 3 washes with cold PBS after replacing the medium with DCFH-DA (25 mol/L). The fluorescence intensity was assessed using a Zeiss fluorescence confocal microscope LSM 800 (Oberkochen, Germany). The ZEN software was used to analyze the images. To perform quantitative flow cytometry (FC500; Beckman Coulter, Brea, CA) analysis, adherent cells were treated with reagents and fluorescent probes, followed by suspension in 500 µl PBS. Measurement and analysis of fluorescence intensity were performed with Cxp (FC500; Beckman Coulter).