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

Authors
  • Eller, Jared M1
  • Stewart, Melissa L2
  • Slepian, Alexandria J2
  • Markwardt, Sheila3
  • Wiedrick, Jack3
  • Cohen, Michael S4
  • Goodman, Richard H2
  • Cambronne, Xiaolu A1
  • 1 Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas.
  • 2 Vollum Institute, Oregon Health & Science University, Portland, Oregon.
  • 3 Biostatistics and Design Program, Oregon Health & Science University, Portland, Oregon.
  • 4 Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon.
Type
Published Article
Journal
Current protocols in cytometry
Publication Date
Apr 01, 2019
Volume
88
Issue
1
Identifiers
DOI: 10.1002/cpcy.54
PMID: 30556645
Source
Medline
Keywords
Language
English
License
Unknown

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. © 2018 John Wiley & Sons, Inc.

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