Agilent Seahorse Extracellular Flux Technology

Seahorse XF Analyzers measure rates of mitochondrial oxygen flux and extracellular acidification rate from live intact cells and tissues in a 24 or 96 multi-well plate format.  

Aglient Core Facility official accreditation badgeThis allows for interrogating key cellular functions such as mitochondrial respiration and glycolysis. XF Analyzers perform compound addition and mixing, label-free analytical detection, and automatic measurement of oxygen consumption and extracellular acidification in ‘real time’.

Seahorse XF Analyzers

Seahorse Extracellular Flux Analysis: an overview

The development of XF analysis to assess mitochondrial respiratory function overcomes certain limitations of classic oxygen electrodes and allows for measurement of mitochondrial energetics in up to 24 or 96 samples at any one time with intact, adherent 2D and 3D cells or tissues.

Data acquired with XF analysis allows one to examine changes in oxygen consumption rate due to ATP turnover, proton leak, and electron transfer capacity. Due to the addition of a proton (H+) detection probe, simultaneous measurements of extracellular acidification are also recorded during an XF assay, making it possible to investigate changes in glycolytic as well as oxidative flux.     

Using an XF analyser to assess mitochondrial function in cells permeabilized with agents such as digitonin or saponin, it is also possible to identify site-specific changes in respiration, such as those as a result of damage to components of the mitochondrial electron transport chain or to diminished anaplerotic enzymes. This type of assay is often useful for understanding mechanisms of mitochondrial dysfunction in disease, identifying novel metabolic actions of thiazolidinediones or for understanding effects of target drugs on mitochondrial function.

Measuring mitochondrial function using the Seahorse Extracellular Flux Analyser

The Seahorse XFe extracellular flux analyser simultaneously measures rates of oxygen consumption and extracellular acidification over timed intervals of about 5-8 minutes.

In a typical mitochondrial function assay, cultured cells or tissue adhered to a specific XFe microplate are washed into mitochondrial respiration media with different substrates and exposed sequentially to an ATP synthase inhibitor, protonophore uncoupler, and a mixture of respiratory complex I and III inhibitors.

This type of assay provides significant insight into mitochondrial function by allowing one to measure routine respiration, ATP synthase inhibited proton leak respiration, coupling efficiency, electron transfer capacity and residual oxygen consumption.

As well as mitochondrial function, the Seahorse XFe flux analyser can be used for other applications where a shift between mitochondrial respiration and glycolysis is observed under certain pathological states, for example, cancer, cardiovascular disease, diabetes, obesity, and neurodegenerative disease.

Example protocol for measuring mitochondrial function with the Seahorse XFe24 Analyser using intact cells

Step 1 – Seed Cells
  • Harvest cells and re-suspend to desired concentration.
  • Seed into into XFe24 cell culture microplate in a total volume of 100 μL of growth medium; do not seed cells in background correction wells (A1, B4, C3, D6).
Step 2 – Hydrate Sensor Cartridge
  • Open the Agilent Seahorse XFe24 Flux Assay Kit and remove contents.
  • Place the Sensor Cartridge next to the Utility Plate.
  • Fill each well of the Utility Plate with 1 mL of XF Calibrant.
  • Lower the Sensor Cartridge into the Utility Plate submerging the sensors in XF Calibrant.
  • Place in a non-CO2 37°C incubator overnight. 
Step 3 – Prepare Assay Media
  • Prepare XF assay medium according to protocol
  • Warm appropriate volume of XF assay medium to 37°C in a sterile bottle.
  • pH to 7.4  and leave at 37°C until ready for use
Step 4 – Wash Cells
  • Retrieve the cell culture plate from the cell culture incubator.

  • View cells under the microscope to: 

    • Confirm cell health, morphology, seeding uniformity and purity (no contamination).

    • Ensure cells are adhered, with a consistent monolayer.

  • Wash adherent cells with XF Assay Media.

  • After sufficient washing leave a final volume of 500 μL assay media in each well.

  • Inspect cells under an inverted light optical microscope to ensure that the cells were not disturbed or washed away during this washing step.

  • Place in 37ºC incubator without CO2 for 24 hours.

Step 5 – Prepare respiratory agents and load into sensor cartridge
  • Make dilutions of stock respiratory reagents to required concentrations in XF assay medium
  • Load 75uL of each agent into the injection ports of the XFe24 sensor cartridge as follows:
    • Oligomycin (ATP synthase inhibitor) to port A
    • BAM 15 (uncoupler) to port B
    • Rotenone and antimycin A (mitochondrial respiratory complex I and complex III inhibitors) to port C
  • Inspect each injection port for uniform loading 
Step 6 – Running the XF assay
  • Open the XF wave software
  • Load your protocol
  • When ready load sensor cartridge (hydrated and loaded with compounds) and utility plate into the tray when prompted.
  • Click I’m ready and let the calibration run
  • Calibration running time is between 10-20 minutes.
  • When the calibration is complete open the tray and eject the utility plate.
  • Remove the utility plate and replace with the cell plate.
  • Load the cell plate to initiate equilibration.
  • After completing equilibration, the assay will automatically begin acquiring baseline measurements (as outlined in the instrument protocol).
Step 7 – Analyse assay results
  • Export the data into Microsoft Excel and analyse using your own analysis template. 

This is a basic protocol of how a typical mitochondrial function assay on an XF analyser is run, but there are many other assay combinations that can be designed to investigate a wide range of different metabolic pathways.

To find out specifically how an experiment using XF analysis can support your research please get in touch with us today by emailing

For specific information on the service packages offered for the XFe24 and XFe96 analysers please refer to our  services page.

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