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.
Department of Neurobiology, Harvard Medical School, Cambridge, MA, USA.
Yin Pun
YP
Hung
Gary
G
Yellen
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002138:Calibration; D045744:Cell Line, Tumor; D002470:Cell Survival; D003600:Cytosol / Q000378:metabolism*; D019344:Lactic Acid / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics*; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D015202:Protein Engineering; D019289:Pyruvic Acid / Q000378:metabolism; D059010:Single-Cell Analysis
D008164:Luminescent Proteins; D009243:NAD; D019344:Lactic Acid; D019289:Pyruvic Acid