The most prevalent human diseases in industrialized countries include obesity, type 2 diabetes, cancer, neurodegenerative, and cardiovascular diseases. A common feature of these ailments is dysregulation of cellular energy metabolism. Mitochondria generate 90% of cellular ATP via oxidative phosphorylation and are central to intermediary metabolism, ROS generation, and apoptosis. However, there is a lack of rapid and sensitive assays to measure metabolism in vitro, especially for drug discovery.
To address this, Seahorse Bioscience (www.seahorsebio.com) introduced the XF24 Extracellular Flux Analyzer. This instrument noninvasively quantifies physiological changes in cellular energetics by measuring the two major energy yielding pathways—mitochondrial respiration and glycolysis—in a microplate format. With the analyzer, compounds affecting mitochondrial function and Fatty Acid Oxidation (FAO) can be detected and their EC50 values and kinetics determined.
Extracellular Flux (XF) assays provide comparable performance to biochemical and radioactive methods without the preparation and use of complicated labels or radioactive materials. The XF24 can also be used to detect physiological signaling of receptors, such as GPCRs, ion channels, and receptor tyrosine kinases.
The Seahorse XF24 measures the rate of change of oxygen (Oxygen Consumption Rate, OCR) and proton concentrations (Extracellular Acidification Rate, ECAR) in the media immediately surrounding living cells in a 24-well microplate (Figure 1A). Therefore, a measurement of the analyte fluxes in the media can be used to determine rates of cellular metabolism. Because XF measurements are non-destructive, cells can be profiled over a period of minutes, hours, or days.
The XF24 can simultaneously determine both the oxidative and glycolytic components of cellular bioenergetics in response to metabolic pathway inhibitors (Figure 1B). Untreated A549 cells show a normal basal level of oxidative and glycolytic metabolism. When exposed for 10 minutes to 100 mM of the glycolysis inhibitor 2-deoxyglucose, the cells shift to an almost exclusive oxidative metabolism. Conversely, when incubated for 30 minutes in 1µM of the Complex I inhibitor, rotenone, the cells shift to glycolytic energy production. Lastly, incubation with both 2-DG and rotenone results in a precipitous drop in activity for both energy yielding pathways.