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 Cohen, Richard Goodman, Xiaolu Cambronne

Research output: Contribution to journalArticle

1 Citation (Scopus)

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
DOIs
StateAccepted/In press - Jan 1 2018

Fingerprint

Flow cytometry
Biosensing Techniques
Biosensors
NAD
Flow Cytometry
Availability
Aptitude
Sensors
Hydrolases
Enzymes
Calibration
Fluorescence
Cells

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Histology
  • Medical Laboratory Technology

Cite this

Flow Cytometry Analysis of Free Intracellular NAD+ Using a Targeted Biosensor. / Eller, Jared M.; Stewart, Melissa L.; Slepian, Alexandria J.; Markwardt, Sheila; Wiedrick, Jack; Cohen, Michael; Goodman, Richard; Cambronne, Xiaolu.

In: Current Protocols in Cytometry, 01.01.2018.

Research output: Contribution to journalArticle

Eller, Jared M. ; Stewart, Melissa L. ; Slepian, Alexandria J. ; Markwardt, Sheila ; Wiedrick, Jack ; Cohen, Michael ; Goodman, Richard ; Cambronne, Xiaolu. / Flow Cytometry Analysis of Free Intracellular NAD+ Using a Targeted Biosensor. In: Current Protocols in Cytometry. 2018.
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