Highly Sensitive Metabolic Assay Equipment

November 30, 2015

University of North Carolina at Chapel Hill
Research
Laboratory Technician

Overall

Performance

Ease-of-Optimization

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Company:

Seahorse Bioscience

Product Name:

XF24 Extracellular Flux Analyzer & Prep Station

Catalog Number:

XF24

Our lab conducts research on a number of cancers, and our research often includes an analysis of the metabolic effects of various mutations, compounds, and genotypes upon cell lines. We used the XF24 analyzer to demonstrate a difference in metabolic response by renal cell carcinoma cell lines in response to several MEK inhibitor compounds. In particular, we selected this machine because of its highly sensitive readout and ability to run 24 samples at once.

Experimental Design and Results Summary

Application

Measurement of Oxygen Consumption Rate and Extracellular Acidification Rates in Cell Lines

Starting Material

Various Renal Cell Carcinoma Cell Lines

Protocol Overview

This machine requires an extensive preparation, and each preparation step is described in extensive detail on the Seahorse Bioscience website, so I will only gloss over it. For each final run, you will need to perform multiple optimization runs in order to determine the appropriate seeding density and reagent concentrations. I recommend planning far in advance, as this machine is extremely finicky and requires precise plating and pipetting for clean results. Furthermore, you will need to set up a program to run the machine. If you're running one of the specific assays recommended, it's very simple - just label everything accordingly. Each compound needs to be at 10X before it's injected into the well and diluted 1:10 down to the working concentration. Loading individual cartridges can be tedious, and requires good diagrams with plenty of time to perform each pipetting maneuver. All in all, extensive preparation is required. Any questions that you have will be answered promptly by the support staff, and are likely addressed in the extremely detailed manuals available for download on the Seahorse Bioscience Website.

Tips

Optimize over and over. Average across multiple runs, and run samples in quadruplicate as opposed to triplicate. Also, double-check that everything was injected from the cartridge in each well...also count cells or spec protein in each well afterwards to normalize the data.

Results Summary

This machine generates a graph of Oxygen Consumption Rate (OCR) that should correlate directly with use of the Electron Transport Chain in cells. In addition, it generates a graph of the Extracellular Acidification Rate (ECAR), which should correlate in theory with lactate production within the cells. What's particularly fascinating about the results is that the machine takes multiple readings and allows you to track the cellular response to reagent addition. By doing so, you can determine a number of things about cellular metabolism that tell you a great deal about how cells respond to stress.

Features Summary

Ability to read OCR and ECAR simultaneously is particulary impressive, in addition to using cartridges to inject your own compounds or reagents as needed.

Additional Notes

Plasticware and Reagents are expensive, and sold in packs of odd quantities.

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Summary

The Good

Extremely sensitive and able to elucidate minor differences in cellular metabolism.

The Bad

Requires extensive optimization procedure, and each run could be completely different from the next...

The Bottom Line

If you're willing to put in the time and effort, you can come out with results that elucidate the extremely complex metabolic responses of cells. Money, time, and optimization aside, the final product can give you more information than several other assays at once in publication-ready graph format. This can raise more questions than it answers, in a good way.

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