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.