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Flow Cytometry Analysis of Free Intracellular NAD+ Using a Targeted Biosensor.


ABSTRACT: Flow cytometry approaches combined with a genetically encoded targeted fluorescent biosensor are used to determine the subcellular compartmental availability of the oxidized form of nicotinamide adenine dinucleotide (NAD+ ). The availability of free NAD+ can affect the activities of NAD+ -consuming enzymes such as sirtuin, PARP/ARTD, and cyclic ADPR-hydrolase family members. Many methods for measuring the NAD+ available to these enzymes are limited because they cannot determine free NAD+ as it exists in various subcellular compartments distinctly from bound NAD+ or NADH. Here, an approach to express the sensor in mammalian cells, monitor NAD+ -dependent fluorescence intensity changes using flow cytometry approaches, and analyze data obtained is described. The benefit of flow cytometry approaches with the NAD+ sensor is the ability to monitor compartmentalized free NAD+ fluctuations simultaneously within many cells, which greatly facilitates analyses and calibration. © 2018 by John Wiley & Sons, Inc.

SUBMITTER: Eller JM 

PROVIDER: S-EPMC6422705 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Flow Cytometry Analysis of Free Intracellular NAD<sup>+</sup> Using a Targeted Biosensor.

Eller Jared M JM   Stewart Melissa L ML   Slepian Alexandria J AJ   Markwardt Sheila S   Wiedrick Jack J   Cohen Michael S MS   Goodman Richard H RH   Cambronne Xiaolu A XA  

Current protocols in cytometry 20181217 1


Flow cytometry approaches combined with a genetically encoded targeted fluorescent biosensor are used to determine the subcellular compartmental availability of the oxidized form of nicotinamide adenine dinucleotide (NAD<sup>+</sup> ). The availability of free NAD<sup>+</sup> can affect the activities of NAD<sup>+</sup> -consuming enzymes such as sirtuin, PARP/ARTD, and cyclic ADPR-hydrolase family members. Many methods for measuring the NAD<sup>+</sup> available to these enzymes are limited bec  ...[more]

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