2.5. MitoSOX Assay

MitoSOX™ Red Mitochondrial Superoxide Indicator (Molecular Probes, Invitrogen, USA) is a fluorogenic probe for highly selective detection of superoxide in the mitochondria of live cells. The probe is a DHE derivate, containing triphenylphosphonium group. Once in the mitochondria, the MitoSOX™ Red reagent is oxidized by superoxide and exhibits red fluorescence [71, 72]. The probe is not oxidized by other ROS/RNS, and its oxidation is prevented by superoxide dismutase [72].

Briefly, MitoSOX™ Red was dissolved in DMSO to 5 mM stock solution, which was diluted with Hank's Balanced Salt Solution (HBSS, containing Ca²⁺ and Mg²⁺) to prepare 3 μM MitoSOX™ Red working solution on the day of experiment. 1 mL of cells (1 × 10⁶ cells/mL for nonadhesive cells and 5 × 10⁵ cells/mL for adhesive cells) was collected by centrifugation, and the pellet was resuspended in 1 mL of 3 μM MitoSOX™ Red. The samples were incubated for 30 min at room temperature, protected from light, washed three times with PBS using centrifugation, and finally resuspended in 1 mL of PBS. The fluorescence intensity was detected immediately at λ_ex = 510 nm and λ_em = 580 nm, using a microplate reader (TECAN Infinite® M1000, Austria) or fluorescence confocal microscope (live cell imaging; magnification 60x) (Olympus DP73, Japan).