Quantifying Mitochondrial Superoxide in Neurons

Mitochondrial superoxide was determined by using Mitosox Red (Thermo Fisher Scientific, M36008). Primary cultured hippocampal neurons with a density of 2 × 10⁴ per well on Lab-Tek chamber slides (Nunc, 177,445) were pre-incubated with 2.5 µM Mitosox Red and 400 nM MitoTracker Green or 5 µM DCF for 20 min in an incubator (5% CO₂, 37 °C) followed by a wash with pre-warmed Neurobasal A medium. Pre-stained neurons were treated with Oligomycin A as indicated in the previous section. 20 nM Hoechst (Thermo Fisher Scientific, 33342) was used to label the nucleus in live cells. The images of Mitosox Red were collected on a Nikon confocal microscope with on-stage incubator (5% CO₂, 37 °C). The intensity was subsequently analyzed by using Nikon NIS Advanced Research Elements 4.13.00. Intra-neuronal superoxide was determined by CM-H2DCFDA (DCF, Thermo Fisher Scientific, C6827) staining as previously described^{14 (link),26} . Images of DCF staining were collected on a Nikon inverted microscope. The intensity was subsequently analyzed by using Addgene.