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

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.