Assessment of extracellular acidification using a fluorescence-based assay

James Hynes (1), Conn Carey (1), Sinead Kriwan (1), Catherine Wark (2) (1) Agilent Technologies, (2) BMG LABTECH 10/2011

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. Here we describe a time resolved fluorescence-based assay for the assessment of Extracellular Acidification which provides data on the rate of conversion of pyruvate to lactic acid and is therefore a convenient measure of glycolytic activity.


The following protocol outlines how such measurements can be carried out on standard microtiter plates using the fluorescent pH-sensitive probe, pH-Xtra and ratiometric time resolved fluorescence detection on a BMG LABTECH microplate reader. Data analysis such as calculation of emission lifetime is greatly simplified by the BMG LABTECH's MARS Data Analysis Software that comes with each reader.

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