MitoSOX measurement

MitoSOX™ Red is a derivative of dihydroethidium, which has selective uptake in actively respiring mitochondria. Once in mitochondria, it is oxidized by superoxide anions producing the red-fluorescent product 2-hydroxyethidium [31]. Cells growing on insert membrane were rinsed with warm Hanks buffered saline solution (HBSS) (Gibco), followed by incubation at 37 °C in the dark for 10 min in HBSS containing 5 μM MitoSOX™ Red (Molecular Probes). CAFs growing on the bottom of the insert were harvested by Accutase®, washed two times with HBSS, and suspended in 400 μL HBSS supplemented with 1% (vol/vol) FBS. Measurements of fluorescence were determined using a FACSCalibur™ flow cytometer (BD Biosciences) with a 488 nm excitation laser and 585/42 nm emission filter. At least 10,000 events were recorded per sample. Relative fluorescent intensity was used as measurement of mitochondrial superoxide production. Results were compared to unstained cells (negative control) and cells treated with medium containing 100 μM Antimycin A, a mitochondrial inhibitor that results in elevated superoxide anion levels (positive control). Analysis was performed using FlowJo V8 software.