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Redox metabolism plays a critical role in multiple pathophysiological settings, including oncogenesis and tumor progression. Until recently, however, our knowledge of key redox processes in living systems was limited by the lack of an adequate methodology to monitor redox potential. Nicotinamide adenine dinucleotide, in its reduced (NADH) and oxidized (NAD(+)) forms, is perhaps the most important small molecule in the redox metabolism of mammalian cells. We have previously developed a series of genetically encoded fluorescent sensors allowing for the quantification of intracellular NADH. Here, we present experimental components and considerations that are required to perform a standardized quantification of intracellular NADH based on these probes. Moreover, we present the initial calibration experiments necessary to obtain reliable data from this approach, we detail a protocol to measure intracellular NADH levels in steady-state kinetic experiments, and we provide consideration on the processing of data. Among various applications, this technique is suitable for the study of redox alterations in malignant cells.

Real-Time Assessment of the Metabolic Profile of Living Cells with Genetically Encoded NADH Sensors