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Equipment

XFe 96 Extracellular Flux Analyzer
Seahorse

The Seahorse XF96e 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 96-well microplate.

The XF96e can determine both the oxidative and glycolytic components of cellular bioenergetics in response to metabolic pathway inhibitors.

What type of cells/samples could be analyzed?

  • Adherent or suspension cells (should be immobilised)

  • Isolated beta-cell islets, myofibers

  • Purified mitochondria

  • Bacteria

 

What can be measured?

  • Mitochondrial respiration (basal, maximal (FCCP treatment) and spare respiratory capacity (Rotenone and Antimycin A treatment)

  • Glycolytic function (glycolysis, glycolytic capacity (Oligomycin treatment), glycolytic reserve (2 Deoxy-D-glucose treatment)

  • Fatty acid oxidation

 

Test duration

Total assay time is about 60 to 90 minutes for each assay (cells need to be splited 24h in advance)

The Seahorse assay in detals

Oxygen consumption rate is measured before and after adding inhibitors.

Measurement of oxygen consumption over 6 min is made repeatedly.

Three measurements are made and averaged to provide reliable measurements.

For the first 18 min, total cellular oxygen consumption is measured. Basal respiration can be calculated from this quantity by subtracting non-mitochondrial respiration.

Next oligomycin, an inhibitor of adenosine-5’-triphosphate (ATP) respiration, is added and a measurement of this is made over the next 18 min. This quantity can be subtracted from the total cellular oxygen consumption to determine ATP-linked respiration and non-mitochondrial respiration can be subtracted from this quantity to obtain proton-leak respiration.

Next carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon, a protonophore, is added and a measurement of this is made over the next 18 min. The protonophore collapses the inner membrane gradient by making the inner membrane permeable to protons. This drives the electron transport chain to function at its maximum rate. Subtracting non-mitochondrial respiration from this quantity produces a measure of maximum respiratory capacity.

Finally antimycin A, a complex III inhibitor, and rotenone, a complex I inhibitor, are added to shut down electron transport chain function. The resulting measurement over the next 18 min represents non-mitochondrial respiration, a measurement that can be used together with the other measurements to calculate respiratory parameters. Finally, reserve capacity is calculated by subtracting basal respiration from maximum respiratory capacity.

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