4.8. Mitochondrial ROS Measurements

To determine mitochondrial ROS levels following NMDAR activation cells were incubated with 5 µM MitoSOX for 30 min at 37 °C. This Live-cell permeant fluorogenic superoxide-sensitive dye targeted the mitochondria selectively and rapidly. The fluorescence intensity before and during the experiment was measured at λ Ex/Em: 510/580 nm. The colocalization experiments were performed in the confocal microscope (Nikon C1 Plus) to evaluate the correct location of MitoSOX by comparison with the subcellular mitochondrial marker Mitotracker green and with Hoechst as the nuclear counterstain. The incubation and preparation of the dyes was performed according to manufacturer instructions. The effects of ASX on mitochondrial ROS levels were assayed in SH-SY5Y cells pre-incubated with increasing concentrations of ASX (in µM: 0.0005; 0.001; 0.01; 0.1; 1, 10) in culture medium supplemented with 1% serum for 24 h before NMDA addition. Treatment with NMDA was performed at the time of the experiment; NMDA was added to the extracellular medium as described for the calcium measurements.