title: Real-time assessment of the metabolic profile of living cells with genetically encoded NADH sensors.
abstract: Redox metabolism plays a critical role in multiple pathophysiological settings, including oncogenesis and tumor progression. Until recently, however, our knowledge of key redox processes in living systems was limited by the lack of an adequate methodology to monitor redox potential. Nicotinamide adenine dinucleotide, in its reduced (NADH) and oxidized (NAD(+)) forms, is perhaps the most important small molecule in the redox metabolism of mammalian cells. We have previously developed a series of genetically encoded fluorescent sensors allowing for the quantification of intracellular NADH. Here, we present experimental components and considerations that are required to perform a standardized quantification of intracellular NADH based on these probes. Moreover, we present the initial calibration experiments necessary to obtain reliable data from this approach, we detail a protocol to measure intracellular NADH levels in steady-state kinetic experiments, and we provide consideration on the processing of data. Among various applications, this technique is suitable for the study of redox alterations in malignant cells.
authors.affiliation: Synthetic Biology and Biotechnology Laboratory, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, Shanghai, China.
authors.forename: Yuzheng
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Synthetic Biology and Biotechnology Laboratory, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, Shanghai, China. Electronic address: yiyang@ecust.edu.cn.
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA. Electronic address: jloscalzo@partners.org.
authors.forename: Joseph
authors.initials: J
authors.lastname: Loscalzo
mesh_terms: D000595:Amino Acid Sequence; D015374:Biosensing Techniques; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009243:NAD / Q000032:analysis* / Q000378:metabolism*; D010084:Oxidation-Reduction; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D009243:NAD
keywords: Fluorescence; Glycolysis; Metabolism; Mitochondria; Redox; Tricarboxylic acid cycle