XFp Extracellular Flux Analyzer User Manual
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1. XFp Analyzer Overview
The Seahorse Bioscience XFp Analyzer measures the rate of change of dissolved oxygen and pH in the
media immediately surrounding living cells cultured in a miniplate. Changes in the extracellular media
are caused by the consumption or production of analytes by the cells. Therefore, a sensitive
measurement of the media flux can be used to determine rates of cellular metabolism with precision
and in a completely non-invasive, label-free manner.
A unique feature of XF technology is its ability to make accurate and repeatable measurements in as
little as five minutes. This is accomplished by isolating an extremely small volume (approximately 2
μ
L)
of media above the cell monolayer. Cellular metabolism causes rapid, easily measured changes to the
microenvironment in this small volume.
Typically, a measurement cycle is performed for 6 minutes. The media is gently mixed and the analyte
levels are then measured until the oxygen concentration drops approximately 20-30% and media pH
declines approximately 0.1-0.2 pH units. The measurement is performed using optical fluorescent
biosensors embedded in a disposable cartridge that is placed into the specially designed Seahorse XFp
Cell Culture miniplate.
Baseline metabolic rates are typically measured 3-4 times, and are reported in pmol/min for OCR
(Oxygen Consumption Rate) and in mpH/min for ECAR (Extracellular Acidification Rate). Compound is
then added to the media and mixed, and then the post-treatment OCR and ECAR measurements are
made and repeated. As cells shift metabolic pathways, the relationship between OCR and ECAR
changes.
The XFp Analyzer system includes a bench top analyzer, disposable sensor/compound delivery
cartridges, tissue-culture treated miniplates for seeding cells for analysis, calibration solution, and
Wave software.
Consumable cartridges, miniplates and calibration reagents are packaged and sold in sets under the
trade name “FluxPaks”.
Assay kits and reagents for performing gold standard XF assays are available. The XFp Cell Mito Stress
Test and XFp Glycolysis Stress Test kits contain pre-measured, verified compounds for assessing the two
major energy pathways in cells and are recommended for characterizing the metabolic phenotype of
cells using the XFp Analyzer.
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