Article

Frex and FrexH: Indicators of metabolic states in living cells

Synthetic Biology and Biotechnology Laboratory, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, Shanghai, China.
Bioengineered bugs 05/2012; 3(3):181-8. DOI: 10.4161/bbug.19769
Source: PubMed

ABSTRACT Reduced nicotinamide adenine dinucleotide (NADH) and its oxidized form play central roles in energy and redox metabolisms. For many years, researchers have relied on the weak NADH endogenous fluorescence signal to determine the NADH level in living cells. We recently reported a series of genetically encoded fluorescent sensors highly specific for NADH. These sensors allow real-time, quantitative measurement of this significant molecule in different subcellular compartments. In this study, we provide a more detailed discussion of the benefits and limitations of these genetically encoded fluorescent sensors. These sensors are utilized in most laboratories without the need for sophisticated instruments because of their superior sensitivity and specificity. They are also viable alternatives to existing techniques for measuring the endogenous fluorescence of intracellular NAD(P)H.

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Available from: Yuzheng Zhao, May 10, 2014
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    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.
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