Understanding abnormal mitochondrial function in pathophysiology has expanded beyond obvious disorders such as diabetes and obesity into those that include aging, cancer, cardiomyopathy, and neurodegeneration. A common component of these ailments is dysregulation of cellular energy metabolism. Since mitochondria generate the vast majority of cellular ATP via oxidative phosphorylation, they have come under closer scrutiny and, in many indications, are being considered as potential targets for clinical intervention.
In addition to ATP production, mitochondria are responsible for the ß-oxidation of short-, medium-, and long-chain fatty acids as well as central to intermediary metabolism, ROS generation, and apoptosis. Hence, measurement of mitochondrial bioenergetics would provide valuable insight into several disorders and possibly lead to identification of targets for drug discovery.
Traditionally, mitochondrial function has been assessed with Clark-type electrode probes for measuring oxygen consumption, luminescent ATP assays for quantification of total energy metabolism, and MTT or Alamar Blue for determination of metabolic activity.
The Clark electrode provides valuable kinetic information but introduces artifact by its continuous consumption of oxygen, presenting a decreasing oxygen pressure to the cells or isolated mitochondria in the measurement chamber. Although oxygen consumption is a good indicator of mitochondrial function, it only measures one component of cellular bioenergetics and therefore the investigator is oblivious to other pathways that contribute to bioenergetic equilibrium, namely glycolysis.
ATP assays are extremely sensitive but they are not an ideal metric of mitochondrial function as cells strive to maintain a particular ATP budget and will adjust metabolism accordingly. Thus, alterations in ATP levels are usually only detectable during pathophysiological changes.
Artifact has been reported from residual ATP present in dying or dead cells. ATP assays are also destructive and lack kinetic information. Perhaps the two greatest deficiencies of ATP assays are that they do not measure ATP turnover and they cannot determine the relative contribution of energy yielding pathways to total ATP yield.
MTT/XTT and Alamar Blue assays are not as sensitive as ATP assays and have been reported to introduce error through cell toxicity, the very parameter they are supposed to be measuring. Both assays are destructive and lack kinetic information.
Seahorse Bioscience (www.seahorsebio.com) recently introduced a new, label-free, assay system—the XF24 Extracellular Flux Analyzer. Extracellular flux (XF) assays measure the two major energy-producing pathways of the cell simultaneously—mitochondrial respiration (oxygen consumption) and glycolysis (extracellular acidification)—in a sensitive microplate format. XF assays work with adherent cells offering a physiologically relevant, real-time cellular bioenergetic assay.
XF assays also provide comparable performance to biochemical and radioactive methods, with better throughput and without the preparation and use of labels or radioactive materials. Thus, the XF assay format overcomes a lot of the problems and deficiencies of traditional assays that either directly or indirectly measure mitochondrial function. In this tutorial, we will explain how bioenergetic measurements are made using XF and show examples of how it is employed in research and drug discovery.