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Agilent’s Seahorse Extracellular Flux Analyzer

Agilent’s Seahorse Extracellular Flux Analyzer

The functionality, health, and performance of skeletal muscle depends on its production and use of energy, mainly in the form of adenosine triphosphate (ATP). Many external and intrinsic factors, including muscle cell fiber type, exercise, age, energy demand, available energy sources, cell type, and disease state, can affect the bioenergetic phenotypes of cells. At Genea we’ve recently begun experiments to determine the changes in metabolism that occur during muscle development in normal and diseased contexts using our myogenic differentiation platform and Agilent’s Seahorse Extracellular Flux Analyzer. With this assay system we can probe a cell type’s basal energy preference phenotype and determine whether it most prefers using mitochondrial respiration or glycolysis to generate ATP. Then, we use metabolic stressor compounds to force cells to maximize their glycolytic rate and to depolarize their mitochondria, yielding an energetic phenotype under conditions of induced energy demand. The comparison of the basic energetics phenotype and stressed phenotype together reveal the cells’ metabolic potential. After establishing the metabolic potential we can delve deeper into potential differences within each energetics pathway.
For instance, in disease contexts where we identify or suspect defects in mitochondrial respiration and function we employ the Cell Mito Stress Test. Here we apply pharmacological modulators of mitochondrial respiration components in order to determine the cells’ basal respiration, ATP production, maximal respiration, and spare respiratory capacity. By breaking down the electron transport chain into individually probed segments we are learning about why and how mitochondrial dysfunction can contribute to both muscle disease pathology and normal myogenesis. On the other hand we can also measure glycolytic function in cultured cells. By using extremely sensitive probes to measure the extracellular acidification rate of our cells’ culture media, the Glycolysis Stress Test measures the conversion of glucose to pyruvate and subsequently lactate, which yields a net extrusion of protons and thereby acidifies the environment. If we run these measures in the presence of drugs that inhibit mitochondrial ATP production we can force cells to switch their bioenergetics profile toward exclusively glycolysis, measure their glycolytic rate, and determine their glycolytic capacity. We are excited to use these powerful Seahorse technologies to better understand the bioenergetic demands of normal myogenic differentiation and to identify novel energetic phenotypes of muscle disease.

 

 

 

 

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