4.5. Mitochondrial Stress Assay

Mitochondrial function was determined by measuring oxygen consumption rate (OCR) of each cell line using XF Cell Mito Stress Test Kit (Agilent Technologies, Santa Clara, CA, USA). FTC133, BCPAP and TT cells transfected with shCOX4 and control cells were seeded in an XF96 cell culture microplate. The sensor cartridge and base medium were prepared by adding 1 mM pyruvate, 2 mM glutamine and 10 mM glucose and stored as per the manufacturer’s instructions. Seahorse assay was run in XF96 Extracellular Flux Analyzer (Agilent Technologies).

Following three baseline OCR measurements, cells were exposed sequentially to oligomycin (0.5 μM), carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) (1 μM), and rotenone/antimycin A (0.5 μM). Oligomycin inhibits ATP synthase (complex V), and the decrease in OCR following injection of oligomycin correlates to the mitochondrial respiration associated with cellular ATP production. FCCP is an uncoupling agent that collapses the proton gradient and disrupts the mitochondrial membrane potential, allowing cells to achieve maximal oxygen consumption rate. As a result, electron flow through the ETC (electron transfer chain) is uninhibited, and oxygen is maximally consumed by complex IV. The FCCP-stimulated OCR can then be used to calculate spare respiratory capacity, defined as the difference between maximal respiration and basal respiration. Spare respiratory capacity is a measure of the ability of the cell to respond to increased energy demand. The third injection is a mix of rotenone, a complex I inhibitor, and antimycin A, a complex III inhibitor. This combination shuts down mitochondrial respiration and enables the calculation of nonmitochondrial respiration driven by processes outside the mitochondria.

Three measurements were recorded after every injection. To normalize the results, cell number/well was quantified using the Celígo Imaging Cytometer (Nexcelom, Lawrence, MA, USA). The assay results were analyzed using Wave program 2.3.0 (Seahorse Bioscience, Billerica, MA, USA).