In vitro analysis of cell metabolism using a long-decay pH-sensitive lanthanide probe and extracellular acidification assay

Metabolic perturbations play a critical role in a variety of disease states and toxicities. Therefore, knowledge of the interplay between the two main cellular ATP generating pathways, glycolysis and oxidative phosphorylation, is particularly informative when examining such perturbations. Here we describe a new fluorescence-based screening assay for the assessment of glycolytic flux and demonstrate the value of such analysis in assessing the cellular "energy budget." The assay employs a long-decay pH-sensitive lanthanide probe to monitor extracellular acidification (ECA) in standard 96- or 384-well plates on a time-resolved fluorescence plate reader. The simple mix-and-measure procedure and fluorescence lifetime-based pH sensing allow the use of standard adherent cell culture techniques, providing high sample throughput and excellent assay performance. The assay also facilitates multiplexed or parallel analysis with existing oxygen consumption and ATP assays, thereby providing a detailed multiparametric assessment of cell metabolism. Data on cellular CO₂ production can also be obtained by comparing sealed and unsealed samples. The utility of the approach in assessing perturbed cell metabolism is demonstrated using a panel of metabolic effectors with known mechanisms of action. More complex metabolic stimuli, such as G protein-coupled receptor (GPCR) activation and perturbed ion homeostasis, are also examined.