MitoXpress-Xtra allows convenient, microplate-based analysis of mitochondrial function through oxygen consumption quantification.

Dr Martin Mangold Dr Martin Mangold

Mitochondrial dysfunction is implicated in numerous disease states and is also a major mechanism of drug-induced toxicity. Oxygen consumption is one of the most informative and direct measures of mitochondrial function. Traditional methods of measuring oxygen consumption are hampered by the limitations of low throughput and high complexity.

Figure 1: Different levels of oxygen consumption displayed by cells treated with antimycin and FCCP as compared to untreated cells

Microplate-based analysis of mitochondrial function

The MitoXpress®-Xtra assay from Agilent Technologies solves these limitations by allowing convenient, plate-based analysis of mitochondrial function. The assay employs MitoXpress®, one of a family of phosphorescent oxygen-sensitive probes developed by Agilent. This time-resolved fluorescence assay is based on the ability of O2 to quench the excited state of the MitoXpress® probe. As the test material respires (e.g., isolated mitochondriacell populationssmall organismstissues, and enzymes), O2 is depleted in the surrounding solution/environment, which is seen as an increase in probe signal. Changes in oxygen consumption reflecting changes in mitochondrial activity are seen as changes in the MitoXpress® probe signal over time.

Figure 2: Changes in environmental oxygen and their effect on intracellular oxygen concentration

The assay is non-chemical and reversible; a decrease in oxygen consumption (an increase in O2 levels) is seen as a decrease in probe signal. MitoXpress® can be analyzed using VANTAstar®CLARIOstar® Plus, and Omega series fluorescence plate readers in Time-Resolved Fluorescence mode, using standard 96- and 384-well microplates. The MitoXpress®-Xtra assay combines the high data quality and information content of the oxygen electrode approach, with the throughput and convenience of microplate-based assays. These capabilities make MitoXpress®-Xtra the ideal tool for rapid compound screening, IC50 generation, and the application of structure-activity relationship approaches.

Examples on the detection of the MitoXpress®-Xtra assay on BMG LABTECH plate readers are discussed in the application notes: Real-time monitoring of intracellular oxygen using MitoXpress-Intra, Real-time measurement of intracellular O2 in mammalian cells, Measuring changes in cellular metabolism by monitoring extracellular acidification and oxygen consumption in real-time and The CLARIOstar with ACU exposes cells to ischemia-reperfusion conditions and monitors their oxygenation.



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