Agilent Technologies Seahorse XFe, Operating Manual

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Introduction

Instrument Overview and Intended Use

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Seahorse XFe Analyzer Operating Manual

Instrument Overview and Intended Use

The Agilent Seahorse XFe analyzer measures the rate of change of dissolved 
oxygen and pH in the media immediately surrounding living cells cultured in a 
microplate. 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 great 
sensitivity and in a totally non-invasive, label-free manner.

A unique feature of the Agilent Seahorse XF technology is its ability to make 
accurate and repeatable measurements in as little as five minutes. The 
instrument, working with a sensor cartridge, isolates a few µL of media above the 
cell monolayer. Cellular metabolism causes rapid, easily measured changes to 
the “microenvironment” in this small volume.

An XFe 

sensor cartridge

 is required to run an assay. The cartridge has 24 or 96 

probes, and each probe has a single multifluor sensor spot that is sensitive to 
both oxygen and proton concentration. The system measures the concentration 
of each analyte over time and automatically calculates the oxygen consumption 
rate (OCR) and extracellular acidification rate (ECAR) simultaneously in every 
well of the microplate.

Typically, a measurement cycle is performed for 5-8 minutes. The media is 
gently mixed, the probe is positioned 200 µm above the well bottom, and the 
analyte levels are measured until the oxygen concentration drops approximately 
20-30% and media pH declines approximately 0.1-0.2 pH units. 

Baseline metabolic rates are typically measured 3-4 times, and are reported in 
pmol/min for OCR and in mpH/min for ECAR, compound is 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 XFe system, comprising a bench top analyzer and touch screen controller, is 
driven by 

Wave software

. This software enables all aspects of Seahorse assays 

including assay setup, instrument control, and data analysis.

Consumables are sold separately and include FluxPaks (comprising sensor 
cartridges, cell plates, and calibrant) as well as a variety of Assay Kits, Reagents, 
and Media. XFe Sensor Cartridges are specific for the instrument type and can 
be purchased exclusively from Agilent.