Flow Cytometry Analysis of Free Intracellular NAD+ Using a Targeted Biosensor. Issue 1 (17th December 2018)

• Record Type: Journal Article
• Authors: Eller, Jared M.; Stewart, Melissa L.; Slepian, Alexandria J.; Markwardt, Sheila; Wiedrick, Jack; Cohen, Michael S.; Goodman, Richard H.; Cambronne, Xiaolu A.
• Editors: Robinson, J. Paul; Darzynkiewicz, Zbigniew; Dobrucki, Jurek; Hyun, William C.; Nolan, John P.; Orfao, Alberto; Rabinovitch, Peter S.
• Is Part Of: Current protocols in cytometry. Volume 88:Issue 1(2019)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2018-12-17
• Subjects: ARTD -- CD‐38 -- circularly permutated fluorescent protein -- fluorescent sensor -- genetically encoded biosensor -- metabolite -- NAD+ -- nicotinamide adenine dinucleotide -- PARP -- sirtuin; Cytology -- Laboratory manuals; Flow cytometry -- Laboratory manuals; Cell separation -- Laboratory manuals; Molecular biology -- Laboratory manuals; Flow Cytometry -- methods; Image Cytometry -- methods; Cell Separation -- methods; Cytological Techniques; Molecular Biology -- methods; Cell separation; Cytology; Flow cytometry; Molecular biology; Laboratory Manuals; Laboratory manuals; 571.6
• DOI: 10.1002/cpcy.54
• Languages: English
• ISSNs: 1934-9297

Abstract

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.