Seahorse Analyzer

Providing the Best Tools... Achieving the Best Research


Photo of Seahorse XFe96 Flux Analyzer

Seahorse XFe96 Flux Analyzer


The two major cellular energy-yielding pathways, mitochondrial respiration and glycolysis, can be measured simultaneously and in real-time using the Seahorse XFe96 instrument. Changes in these two energy pathways affect the immediate extracellular environment, and the resulting changes in O2 concentration (oxygen consumption or OCR), and pH (extracellular acidification rate, or ECAR) can then be measured using this technology.


  1. The two major metabolic pathways are measured simultaneously and in real-time
  2. Experiments are performed with living cells in multi-well plates
  3. A large variety of cell types can be analyzed: primary cells, established cell lines, spheroids, isolated mitochondria, and small tissue pieces
  4. The cells and plates are not affected by the measurements and may be used for additional assays
  5. Experiments can be performed using adherent or suspension cells (suspension cells require an additional attachment step)
  6. Only a small number of cells is required (4,000 – 500,000 per well)
  7. Measurements can be repeated multiple times for kinetic analysis
  8. Measurements can be taken for minutes, hours, or even days
  9. The technology is completely non-invasive and does not require the addition of dyes, labels or reporters
  10. Up to four test compounds (drugs or substrates) can be added automatically to each well, and measurements are taken before and after addition of the substance
  11. The software required for analysis is free (available for PCs only)
  12. Average instrument run time is 3-5 hours

The Agilent Seahorse analyzers utilize patented transient micro-chambers and embedded fluorophores to make rapid, sensitive, and precise metabolic measurements in real time. The key to the XF assays is the disposable assay kit, which consists of a cell culture plate and a disposable biosensor cartridge. Cells (or other samples) are plated in the cell culture plate and the sensor cartridge is placed above it, then both are inserted into the instrument. Two fluorophores embedded in polymer at the bottom of the cartridge sleeve function as biosensors. One fluorophore is quenched by oxygen and measures mitochondrial metabolism, while the other fluorophore is sensitive to protons and indicates glycolysis. Fiber optic bundles are simultaneously lowered into each cartridge sleeve and emit light, resulting in excitation of the embedded fluorophores. Changes in fluorescence emission due to alterations in oxygen and proton levels are then detected by the sensors.

The rapid, real time measurements are made possible because the fiber optic bundles are lowered to 200 µm above the cell layer. This creates a transient micro-chamber and allows for immediate measurements. Once the sensors detect a 10% change in oxygen or a similar change in proton levels, the sensors rise and the cells are restored to normal levels. This process can be repeated many times to generate kinetic data. In addition, more data can be collected after the addition of up to 4 compounds directly into the well. Measurements can be taken before and after the injection of inhibitors, stimulators, and substrates to gain more knowledge about the metabolic pathways.

A mandatory training session is required before an operator is allowed to use the instrument. An initial meeting is scheduled to discuss the various steps involved in setting up/running an experiment as well as the objectives of the experiment. General information will be provided by email in advance, and the initial meeting takes approximately one hour. The training fee includes the initial consultation and the instrument fee for the first experiment. For questions about the instrument or to set up a meeting, please contact Kathy Schreiber.

Training Fee: $100

Cost per experiment (per day): $100

XFe96 Flux packs are sold individually at the CIC (when available). This includes a disposable sensor cartridge, cell culture microplate, calibration plate, and XF calibrant. $75.00

Seahorse consumable items can be ordered through the DNA Core.