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Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Flow Cytometry Analysis of Free Intracellular NAD + Using a Targeted Biosensor.
Current Protocols in Cytometry 2019 April
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.
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