2.6. Measurement of Reactive Oxygen Species (ROS) Production

SH-SY5Y cells were seeded in 96-well plates and pretreated with various concentrations of GH (dissolved in PBS). After incubation for 1 h at 37°C, scopolamine was cotreated with GH for 1 h. Afterwards, 30 μM DCFH-DA was added to the culture medium for 30 min at 37°C. The fluorescence of DCF, which is the oxidation product of DCFH-DA, was measured (excitation (Ex)/emission (Em) = 485/535 nm) using a VICTOR X3 plate reader (Perkin Elmer, Waltham, MA, USA) [30].