NADH is an essential redox cofactor in numerous metabolic reactions, and the cytosolic NADH-NAD(+) redox state is a key parameter in glycolysis. Conventional NADH measurements rely on chemical determination or autofluorescence imaging, which cannot assess NADH specifically in the cytosol of individual live cells. By combining a bacterial NADH-binding protein and a fluorescent protein variant, we have created a genetically encoded fluorescent biosensor of the cytosolic NADH-NAD(+) redox state, named Peredox (Hung et al., Cell Metab 14:545-554, 2011). Here, we elaborate on imaging methods and technical considerations of using Peredox to measure cytosolic NADH:NAD(+) ratios in individual live cells.
Stangherlin, A., Koschinski, A., Terrin, A., Zoccarato, A., Jiang, H., Fields, L. A., & Zaccolo, M. (2014). Fluorescent Protein-Based Biosensors, 1071, 59–71. https://doi.org/10.1007/978-1-62703-622-1