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.