How XF Technology Works
How Extracellular Flux is Measured
The XFe Analyzer measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) at intervals of approximately 2-5 minutes. OCR is an indicator of mitochondrial respiration, and ECAR is predominately the result of glycolysis.
Real-time measurements of OCR and ECAR are made by isolating an extremely small volume (less than 7µl) of medium above a monolayer of cells within a microplate. Cellular oxygen consumption (respiration) and proton excretion (glycolysis) causes 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 continues to measure the concentrations until the rate of change is linear and then calculates the slope to determine OCR and ECAR, respectively.
Once a measurement is completed, the probes lift which allows the larger medium 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 drugs 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 two or four drugs may be added to the assay cartridge for use during the assay.
The assay cartridge is first placed into the XFe 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 twice before adding inhibitors, stimulants, substrates, or compounds into the drug delivery chambers. OCR is reported in units of nmoles/minute and ECAR in mpH/minute. Compounds that have been preloaded into the drug delivery chambers of the assay cartridge are then pneumatically injected, sequentially, into the media in each well. After mixing, post-exposure OCR and ECAR measurements are made multiple times.
Because XF measurements are non-destructive, the metabolic rate of the same cell population can be measured repeatedly over time while up to two or four different drugs can be injected sequentially into each well. Upon completion of an XF assay, other types of biological assays such as cell viability can be performed on the same plate. Total assay time is typically 35 to 90 minutes. To maintain normal cell physiology, a temperature control system maintains the XFe Analyzer's internal environment at 37° C.
XF Stress Test Kits & Reagents
Complete, easy-to-use XF Stress Test Kits and reagents are available that help simplify cellular bioenergetics by providing pre-calibrated, pre-tested reagents for measuring mitochondrial respiration, glycolysis and fatty acid oxidation in cells.
Instrument Sensitivity: The Results Speak for Themselves.
XF technology has exceeded the performance of any previous non-invasive metabolic readout. Measurements of cellular metabolism, from one well to the next, one plate to the next, or one day to the next, typically produce CVs of less than 20%. Measurements of the change in cellular behavior as a result of compounds addition typically produce c.v.'s of less than 10%, and changes of as little as 5% can often be seen with replicate wells.
Next Seahorse Webinar
Metabolic Adaptations in Fumarate Hydratase Deficient Cells
Christian Frezza, PhD,
Hutchison/MRC Research Centre, Cambridge, UK
May 29, 2013
Advanced XF Training
Understanding When and How to Use Intact Cells and Isolated Mitochondria in Investigating Bioenergetics
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XF Stress Test Kits
XF Glycolysis Stress Test Kit
Now it's easy to measure glycolytic function in cells.
Learn MoreXF Cell Mito Stress Test Kit
Everything you need to measure mitochondrial function in cells.
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