Simultaneous monitoring of intracellular ATP and oxygen levels in chondrogenic differentiation using a dual‐color bioluminescence reporter

Abstract

A number of assay methods which measure cellular metabolic activity have only measured intracellular ATP levels because it has been speculated that ATP production and oxygen consumption are obligatorily coupled to each other under normal conditions. However, there exist many cases in which ATP production and oxygen consumption are uncoupled. Therefore, measurement of only intracellular ATP levels has a limit for understanding the overall metabolic states during various cellular functions. Here, we report a novel system for simultaneously monitoring intracellular ATP and oxygen levels using a red-emitting Phrixothrix hirtus luciferase (PxRe) and a blue-emitting Renilla luciferase (Rluc). Using this system, we monitored the dynamic changes in both intracellular ATP and oxygen levels during chondrogenesis. We found that the oxygen level oscillated at twice the frequency of ATP in chondrogenesis and the oxygen oscillations have an antiphase mode to the ATP oscillations; we also found an independent mode for the ATP oscillations. This result indicates that both mitochondrial and non-mitochondrial respiration oscillate and thus play a role in chondrogenesis. This dual-color monitoring system is useful for studying metabolic regulations that underlie diverse cellular processes.