Mitochondrial superoxide assay

Mitochondrial superoxide production in response to Aβ_1-42 exposure was quantified using MitoSOX Red (Cat. No. {"type":"entrez-nucleotide","attrs":{"text":"M36008","term_id":"214108","term_text":"M36008"}}M36008 (2019), Molecular Probes) according to the manufacturer’s guidelines. Cerebrovascular endothelial cells were seeded on 96-well plates at a density of 1.5x10⁴ cells per well. Endothelial cells were allowed to proliferate for 24 h ( 85% confluency). Cells were rinsed twice with pre-warmed DMEM and then exposed to 0-, 5-, 9-, and 18-μM Aβ_1-42 for 24 h. After exposure to Aβ_1-42, cells were rinsed 3 times with warm DMEM+and endothelial cells were loaded with 5 μM MitoSOX Red diluted in DMEM+for 10 min at 37°C. After incubation, cells were rinsed 3 times with warm DMEM+. MitoSOX fluorescence was measured with a spectrophotometer by endpoint kinetic set to an excitation of 510 nm and an emission of 580 nm. Each well was measured in relative fluorescence units (RFU). The RFU measurement from each well per group (n =12) was normalized to the average RFU of the control (0 μM Aβ_1-42) group to produce a fold-change value.