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

Jared M. Eller, Melissa L. Stewart, Alexandria J. Slepian, Sheila Markwardt, Jack Wiedrick, Michael S. Cohen, Richard H. Goodman, Xiaolu A. Cambronne

Research output: Contribution to journalArticle

1 Scopus citations

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.

Original languageEnglish (US)
Article numbere54
JournalCurrent Protocols in Cytometry
Volume88
Issue number1
DOIs
StatePublished - Apr 2019

Keywords

  • ARTD
  • CD-38
  • NAD
  • PARP
  • circularly permutated fluorescent protein
  • fluorescent sensor
  • genetically encoded biosensor
  • metabolite
  • nicotinamide adenine dinucleotide
  • sirtuin

ASJC Scopus subject areas

  • Biochemistry
  • Histology
  • Medical Laboratory Technology

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  • Cite this

    Eller, J. M., Stewart, M. L., Slepian, A. J., Markwardt, S., Wiedrick, J., Cohen, M. S., Goodman, R. H., & Cambronne, X. A. (2019). Flow Cytometry Analysis of Free Intracellular NAD+ Using a Targeted Biosensor. Current Protocols in Cytometry, 88(1), [e54]. https://doi.org/10.1002/cpcy.54