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

Flow Cytometry Analysis of Free Intracellular NAD+ Using a Targeted Biosensor. Curr Protoc Cytom. 2018 Dec 17;:e54 Authors: Eller JM, Stewart ML, Slepian AJ, Markwardt S, Wiedrick J, Cohen MS, Goodman RH, Cambronne XA 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. PMID: 30556645 [PubMed - as supplied by publisher]
Source: Current Protocols in Cytometry - Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research