Mitochondrial Assays in SH-SY5Y Cells

Cell-based mitochondrial activity assays were performed using SH-SY5Y cells in 96-well black plates with clear bottom as described previously in detail^{22 (link),47 (link)}. The assays cover the quantitative assays for cell viability (calcein), complex 1, methyl thiazyl tetrazolium-mitochondrial dehydrogenase activity (MTT), mitochondrial membrane potential (TMRE, Molecular Probe, Eugene, OR, USA), ATP contents, and CM-H₂DCFDA- or MitoSox (Molecular Probe)-dependent reactive oxygen species (ROS). The fluorescence intensities at 550 nm/580 nm for TMRE, at 510 nm/580 nm for Mito Sox, or 494 nm/522 nm for DCF-DA were normalized by Hoechst intensity at 355 nm/480 nm (Spectramax Gemini EM, Molecular Devices, Sunnyvale, CA, USA). The Caspase-Glo3/7 assay system (Promega Co, Madison, WI, USA) was used for caspase detection according to the manufacturer’s instructions. Briefly, the drug-pretreated SH-SY5Y cells were exposed to 1 mM MPP⁺ for 24 h, and subsequently incubated with the Caspase-Glo reagent containing proluminescent DEVD tetrapeptide substrate for 1 h. The luminescent intensity was measured by a luminometer (Lumat LB 9501/16, Berthold, Wildbad, Germany).