The pH-Xtra assay is a TRF-based assay to assess extracellular acidification as a measure of glycolytic activity.

Dr Ann-Cathrin Volz Dr Ann-Cathrin Volz

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.

Extracellular acidification assessment

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. 

Fig.1: example of extracellular acidification curve

The pH-Xtra assay

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.

Microplate-based detection

Examples on the detection of the pH-Xtra assay assay on BMG LABTECH plate readers are discussed in the application notes: Measuring changes in cellular metabolism by monitoring extracellular acidification and oxygen consumption in real-time, Measuring mitochondrial function and glycolytic flux in 3D cell cultures and Assessment of extracellular acidification using a fluorescence-based assay.



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