Live-Cell Imaging of Cytosolic NADH–NAD+ Redox State Using a Genetically Encoded Fluorescent Biosensor

  • Yin Pun Hung
  • Gary Yellen
Part of the Methods in Molecular Biology book series (MIMB, volume 1071)


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.

Key words

NADH Glycolysis Lactate dehydrogenase Sensor calibration Single cell imaging 



We thank Mathew Tantama for careful reading of this manuscript. This work was supported by the Albert J. Ryan fellowship, the Stuart H.Q. and Victoria Quan predoctoral fellowship in neurobiology (both to Y.P.H.), and the U.S. National Institutes of Health (R01 NS055031 to G.Y.).


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Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Yin Pun Hung
    • 1
  • Gary Yellen
    • 1
  1. 1.Department of NeurobiologyHarvard Medical SchoolCambridgeUSA

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