Measuring Intracellular Superoxide Anion

MitoSOX™ Red (Invitrogen, Burlington, ON, Canada) was used to estimate intracellular superoxide anion production as already described [42 (link),43 (link),46 (link)]. Neuronal PC12 cells were seeded at 25,000 cells/cm², differentiated and treated on collagen-coated circular glass coverslips. After treatment, NGF-differentiated PC12 cells were washed with Hank’s buffered salt solution (HBSS) and incubated for 10 min at 37 °C with a 5 µM solution of MitoSOX™ Red. Nuclei were counterstained with Hoescht 33342 (5 μg/mL) for 15 min at 37 °C, and then cells were fixed with 4% paraformaldehyde (PFA), mounted on glass slides with Prolong Antifade kit (Invitrogen), examined under a Leitz Orthoplan fluorescence microscope (Leica, Wetzlar, Germany) and photographed with a QImaging camera (Nikon, Mississauga, ON, Canada). Fluorescence intensity was measured using NIS Elements 2.2 software (Nikon, Mississauga, ON, Canada).