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How Agilent Seahorse XF Analyzers Work

How Seahorse Analyzers Measure Extracellular Flux

Seahorse XFe Analyzer 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.
Transient Microchamber
Transient Microchamber
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.
Compound Injection Ports
Compound Injection Ports
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

XF Kits
Complete, easy-to-use 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.

Don't Take Our Word For It

Since its introduction in 2006, Seahorse XF technology has been used in over 2500 peer-reviewed publications. 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.
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For Research Use Only. Not for use in diagnostic procedures.