Use of genetically encoded sensors to monitor cytosolic ATP/ADP ratio in living cells

ATP is not only recognized as the universal energy "currency" in most cells but also plays a less well-known role as an intracellular and extracellular messenger. Here, we review novel approaches for measuring free ATP (or ATP/ADP ratios) in living mammalian cells by using genetically encoded sensors. We also discuss the key technical aspects of routine real-time ATP/ADP monitoring using as a model one of the last-generation fluorescent probes, a fusion protein commonly known as "Perceval." Finally, we present detailed guidelines for the simultaneous measurement of cytosolic ATP/ADP ratios and Ca^{2 +} concentrations alongside electrical parameters in individual pancreatic β cells, in which energy metabolism is tightly linked to plasma membrane excitability to control the secretion of insulin. With appropriate variations, this approach can be adapted to the study of cytosolic ATP/ADP ratios and Ca^{2 +} concentrations in malignant cells, two important aspects of oncometabolism.