Seahorse XF Analyzers measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live cells in a multi-well plate, 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 OCR and ECAR in real time.
OCR and ECAR rates are key indicators of mitochondrial respiration and glycolysis and these measurements provide a systems-level view of cellular metabolic function in cultured cells and ex-vivo samples.
For more information on choosing the assay that is right for you, visit our
XF Instrument Selection Guide.
How Extracellular Flux is Measured
Agilent Seahorse XF Analyzers
measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) at intervals of approximately 5-8 minutes. OCR is an indicator of mitochondrial respiration, and ECAR is largely the result of glycolysis.
Real-time measurements of OCR and ECAR are made by isolating an extremely small volume (about 2 μL) of medium above a monolayer of cells within a microplate.
Cellular oxygen consumption (respiration) and proton excretion (glycolysis) cause rapid, easily measurable changes to the concentrations of dissolved oxygen and free protons in this "transient microchamber" which are measured every few seconds by solid state sensor probes residing 200 microns above the cell monolayer.
The instrument measures the concentrations for 2-5 minutes then calculates the OCR and ECAR, respectively.
Once a measurement is completed, the probes lift which allows the larger medium volume above to mix with the medium in the transient microchamber, restoring cell values to baseline. An integrated drug delivery system allows sequential addition of up to four compounds per well at user-defined intervals.
How an XF Assay is Performed
Prior to the start of the XF assay, cells are seeded into the wells of an XF cell culture microplate. Seahorse supplied,
bicarbonate-free medium is used during the assay. Up to four drugs may be added to the assay cartridge for automated injection during the assay. The assay cartridge is first placed into the analyzer to allow automatic calibration of optical sensors. Next, the cell culture plate is inserted into the instrument.
Typically, baseline (basal) OCR and ECAR rates are measured three or four times before adding inhibitors, stimulants, substrates, or compounds by way of the drug delivery ports.
OCR is reported in units of pmol/minute and ECAR in mpH/minute. Compounds that have been preloaded into the drug delivery ports of the assay cartridge are then pneumatically injected, sequentially, into the media in each well. After gentle mixing, OCR and ECAR measurements are made multiple times.
Because Seahorse XF measurements are non-destructive,
the metabolic rate of the same cell population can be measured repeatedly
over time while up to four different drugs are injected sequentially into each well. Total assay time is typically 60 to 90 minutes. Upon completion of an XF assay, other types of biological assays such as cell viability can be performed on the same plate. To maintain normal cell physiology, a temperature control system maintains the cellular environment at 37°C (or other pre-set temperature).
Seahorse XF Test Kits & Reagents
Seahorse consumables are developed specifically for use with the Seahorse XF Analyzer to ensure reliability and consistency of results. Seahorse XF kits and reagents help simplify running an XF assay by providing pre-calibrated, pre-tested reagents for measuring mitochondrial respiration, glycolysis and fatty acid oxidation in cells.
Specially formulated, unbuffered media adds ease-of-use, simply add supplements as required by the experiment and adjust the pH. Seahorse XF FluxPaks also simplify your XF assays (and ordering) by providing the correct ratio of assay kits to microplates and calibrant. Seahorse XF microplates have been developed specifically for the Seahorse XF Analyzer to maximize the sensitivity of the sensor cartridge measurements, and provide valuable functional metabolic data from living cells, isolated mitochondria, islets and spheroids.
Seahorse XF test kits and reagents
help simplify cellular bioenergetics by providing pre-calibrated, pre-tested reagents for metabolic phenotyping, mitochondrial respiration, glycolysis and fatty acid oxidation in cells.
How to Run an Assay
Learn how to run an XF assay
The Seahorse XF Assay Learning Center provides protocols, techniques and resources aligned to each step of the XF workflow to ensure optimal assay performance and results. The XF Assay Learning Center has been recently updated to reference the new Seahorse XF Real-Time ATP Rate Assay for performing initial cell characterization.
Since its introduction in 2006, Seahorse XF technology has been used in over 2500
The power and utility of Seahorse XF technology allows researchers in a wide variety of application areas to better understand their cellular models and make ground-breaking discoveries about the critical role of metabolism in a wide variety of diseases.
Additional Educational Resources
New resources are continually being developed to ensure you have the tools you need to be successful in your research area. Browse some of the latest resources to help you enhance your research.
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Representatives are available to demonstrate how Agilent Seahorse XF Analyzers can solve your research needs. Schedule your demo today!