Designed for Scientists by Scientists

How the XF24 Extracellular Flux Analyzer Works

From the beginning, Seahorse Bioscience has worked closely with senior scientists, directors and distinguished research fellows at some of the world's largest pharmaceutical companies including Abbott Laboratories, Amgen, AstraZeneca, Bristol Myers Squibb, GlaxoSmithKline, Harvard, Johnson & Johnson, and Merck & Company.

The vision was to develop a series of non-invasive instruments that enabled scientists to measure cellular bioenergetics in a microplate format - quickly, easily and in real-time.

A testament to the fruition of that vision and utility of the XF Series is the number of different therapeutic groups within each organization that are leveraging this novel technology today.

How Extracellular Flux is Measured

The XF24 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.

Rapid measurements of OCR and ECAR are made by isolating an extremely small volume (less than 10μL) of media above a monolayer of cells within an XF 24-well tissue culture microplate. Cellular oxygen consumption and proton excretion causes rapid, easily measurable changes to the "microenvironment" in this small volume.

Dissolved oxygen and pH levels within the microenvironment are measured every 22 seconds by inert optical biosensors that reside approximately 300 microns above the cell monolayer. Over a period of minutes, cells consume oxygen and secrete protons until the dissolved oxygen concentration drops approximately 10%, and extracellular acid level changes approximately 0.2 mpH units.

OCR and ECAR are determined from the rate of change of oxygen and pH levels within the microenvironment.

Once a measurement is completed, the well is stirred to mix the small volume of media used for measurement with the larger volume stored in the well. Measurements may be repeated indefinitely since no dyes are added.

An integrated drug delivery system allows sequential addition of up to four drugs at user-defined intervals. Varying doses of a single drug may be added to determine the dose response of a single population of cells, or agonist/inhibitor sets may be used for MOA studies.

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 media is used during the assay. Up to four drugs may be added (approx 50uL) to the assay cartridge for use during the assay.

The assay cartridge is first placed into the XF24 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 drug addition. OCR is reported in units of nmoles/minute and ECAR in mpH/minute. Drugs that have been preloaded into the drug delivery chambers of the assay cartridge are then pneumatically injected into the media in each well. After 3-5 minutes of 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 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 XF24 Analyzer's internal environment at 37 degrees Celsius.

Instrument Sensitivity: The Results Speak for Themselves.

As a result of a series of novel inventions, we believe that our 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 c.v.'s of less than 20%. Measurements of the change in cellular behavior as a result of drug addition typically produce c.v.'s of less than 10%. Changes of as little as 5% can often be seen with replicate wells.