Metabolic perturbations play a critical role in a variety of disease states and toxicities. Knowledge of the interplay between the two main cellular ATP generating pathways, glycolysis and oxidative phosphorylation, is therefore particularly informative when examining such perturbations. The Agilent pH-Xtra assay is a time-resolved fluorescence based assay that can assess extracellular acidification which provides data on the rate of conversion of pyruvate to lactic acid, and is therefore a convenient measure of glycolytic activity. Such an assay is particularly informative when assessing alterations in glucose metabolism, detecting glycolytic inhibition and as a confirmatory analysis in the identification of mitochondrial dysfunction.

This assay can be analyzed with the Agilent fluorescent pH-sensitive probe, pH-Xtra, and ratiometric time-resolved fluorescence detection in standard 96- and 384-well microplates. This approach overcomes the calibration and biocompatibility issues associated with some existing probes thereby allowing conventional cell culturing and assay procedures whilst also facilitating accurate quantitative analysis. In addition, spectral compatibility with MitoXpress®, the Agilent oxygen sensitive probe, facilitates a multiplexed measurement approach providing a comprehensive metabolic assessment of test cells.

pH-Xtra assays performing microplate readers

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