Live-Cell Imaging of Cytosolic NADH–NAD+ Redox State Using a Genetically Encoded Fluorescent Biosensor
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 wordsNADH 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.).
- 1.Nicholls DG, Ferguson SJ (2002) Bioenergetics, 3rd edn. Academic, LondonGoogle Scholar
- 2.Avi-Dor Y, Olson JM, Doherty MD, Kaplan NO (1962) Fluorescence of pyridine nucleotides in mitochondria. J Biol Chem 237:2377–2383Google Scholar
- 12.Bücher T, Brauser B, Conze A, Klein F, Langguth O, Sies H (1972) State of oxidation-reduction and state of binding in the cytosolic NADH-system as disclosed by equilibration with extracellular lactate-pyruvate in hemoglobin-free perfused rat liver. Eur J Biochem 27:301–317PubMedCrossRefGoogle Scholar