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

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 appr

SUBMITTER: Eller JM 

PROVIDER: S-EPMC6422705 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature