Agilent Seahorse Extracellular Flux Technology

The development of XF analysis to assess mitochondrial respiratory function overcomes certain limitations of classic oxygen electrodes and allows for measurement of mitochondrial energetics in up to 8, 24 or 96 samples at any one time with intact, adherent 2D and 3D cells or tissues.

Data acquired with XF analysis allows one to examine changes in oxygen consumption rate due to ATP turnover, proton leak, and electron transfer capacity. Due to the addition of a proton (H+) detection probe, simultaneous measurements of extracellular acidification are also recorded during an XF assay, making it possible to investigate changes in glycolytic activity as well as oxidative flux.      

Using an XF analysis to probe mitochondrial function in cells permeabilized with agents such as digitonin or saponin, makes it possible to identify site-specific changes in respiration, such as those as a result of damage to components of the mitochondrial electron transport chain or to diminished anaplerotic enzymes. This type of assay is often useful for understanding mechanisms of mitochondrial dysfunction in disease, identifying novel metabolic actions of thiazolidinediones or for understanding effects of target drugs on mitochondrial function.

The Seahorse XFe extracellular flux analyzer simultaneously measures rates of oxygen consumption and extracellular acidification over timed intervals of about 5-8 minutes.

In a typical mitochondrial function assay, cultured cells or tissue adhered to a specific XFe microplate are washed into mitochondrial respiration media with different substrates and exposed sequentially to an ATP synthase inhibitor, protonophore uncoupler, and a mixture of respiratory complex I and III inhibitors.

This type of assay provides significant insight into mitochondrial function by allowing one to measure routine respiration, ATP synthase inhibited proton leak respiration, coupling efficiency, electron transfer capacity and residual oxygen consumption.

As well as mitochondrial function, the Seahorse XF^e flux analyser can be used for other applications where a shift between mitochondrial respiration and glycolysis is observed under certain pathological states, for example, cancer, cardiovascular disease, diabetes, obesity, and neurodegenerative disease.

*cell type dependant 

To find out howXF analysis can support your research please get in touch with us today by emailing j.p.barlow@bham.ac.uk. 

For specific information on the service packages available on our XFe24 or XFe96 analyzer please refer to our services page.