Measuring the mitochondrial oxygen consumption rate (OCR)

Mitochondrial function was determined by using the XF Cell Mito Stress Test Kit (Seahorse Bioscience, MA, USA) with a Seahorse XFe96 Extracellular Flux Analyzer (Seahorse Bioscience, MA, USA). MCF-7 cells were seeded in a specialized 96-well tissue culture plate (XF96 microplate). Next day TPP derivatives were added and the plate was incubated for 72 hours. Before the experiment media was changed to XF base medium (including 1 mM pyruvate, 2 mM glutamine and 10 mM glucose) and cells were incubated at 37°C in a CO₂-free atmosphere for one hour before measurement. After detection of basal oxygen consumption rate (OCR) (an indicator for mitochondrial respiration) OCR responses were evaluated towards the application of oligomycin (1 µM), FCCP (600 nM), and the combination of antimycin (1 µM) and rotenone (1 µM). From these measurements various parameters of mitochondrial function were determined. To determine cell viability in the measured wells sulphorodamine (SRB) assay was performed. Oxygen consumption rate (OCR) values were then normalized to the given SRB values.