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Methods Mol Biol. 2014;1071:83-95. doi: 10.1007/978-1-62703-622-1_7.

Live-cell imaging of cytosolic NADH-NAD+ redox state using a genetically encoded fluorescent biosensor.

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  • 1Department of Neurobiology, Harvard Medical School, Cambridge, MA, USA.


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.

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