4.8. Mitochondrial Superoxide (MitoSOX) Analysis

Levels of mitochondrial superoxide species (mtROS) were determined by use of the mitochondrial specific fluorescent probe MitoSOX Red (ThermoFisher Scientific, Waltham, MA, USA) in two independent assays; (1) fluorescent microplate reader, and data corroborated with (2) fluorescent microscopy.

In brief, cells were seeded at 30,000 cells/well onto a sterile 96-well black microplate (Corning, Costar) 48 h prior to experimentation. After 24 h media was refreshed, and the cells were treated as previously outlined with an additional control group of cells treated with 1 mM rotenone (Sigma Aldrich) for 2 h at 37 °C and 5% (v/v) CO2(g). Following the incubation period, MitoSOX™ Red reagent was reconstituted in sterile DMSO immediately prior to its use to yield a 5 mM stock solution which was further diluted in sterile HPPS at a final concentration of 5 µM. Subsequent to washing the cells in HBBS, this working solution of MitoSOX™ Red was added onto the cells and the plate incubated for 30 min at 37 °C and 5% (v/v) CO₂(g). The cells were washed 3 times in HBSS, and the fluorescence was recorded at 510/580 nm, immediately and every 15 min over a 60-min period using an Infinite^® M200 PRO Plate reader (Tecan, Männedorf, Germany). Data was recorded and analysed in Microsoft^® Excel^® (2013, v7) and normalised against total cellular protein following BCA protein assay on the same plate.

The experimental design generally followed what was outlined in (1) with cells seeded at 10,000 cells/well and no rotenone treatment was included in the assay. Following optimistaion of dose, MitoSOX™ was added at a final concentration of 1 µM to the cells with incubation and washing cycles as also described in (1). To confirm the MitoSOX™ fluorescent signal was selective for mtROS, the mitochondrial specific fluorogenic dye MitoTracker™ Green FM (ThermoFisher Scientific) was used to demonstrate co-localisaton of signal. MitoTracker™ was added to the cells at final concentration of 150 nM and the plate incubated for 10 min at 37 °C and 5% (v/v) CO₂(g). The cells were then imaged under fluorescent microscopy using a Zeiss Axio Scope A.1 (Carl Zeiss, Melbourne, Australia) under x 20 magnification and appropriate filter sets. The captured images were then analysed using Zen2 Lite software (Carl Zeiss, Melbourne, Australia).