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 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.

Subcellular Characterization of Nicotinamide Adenine Dinucleotide Biosynthesis in Metastatic Melanoma by Using Organelle-Specific Biosensors

“…The biosensor works by decreasing its fluorescence at 488 nm in the presence of increased levels of NAD + , while its 405 nm fluorescence is unaffected by substrate variations and can be used to normalize the biosensor expression levels. Therefore, a decrease in NAD + levels leads to an increase of the 488 nm/405 nm fluorescence ratio (9,15,19). From previous works, it is known that FK866 treatment leads to a significant drop of total intracellular NAD + levels beginning few hours after treatment (26).…”

“…In order to quantify NAD + fluctuations, we generated specific calibration curves for the cytosolic, the nuclear and the mitochondrial biosensors ( Figure 3) (9,15,19). To do so, cells expressing cytosolic and nuclear biosensors were saponin-permeabilized to allow exogenous NAD + (used in a range of concentrations between 0 and 4 mM for cytosol and from 0 to 3mM for nucleus) to enter the cell or nucleus, as previously described (9).…”

“…DNA was amplified, incorporated in lentiviral particles (see below) and used for stable cell transduction. The LigA-cpVENUS biosensor presents two excitation peaks, one at 488 nm that decreases according to NAD + elevation and a second one at 405 nm that is unaffected by substrate variations, serving as internal control and system calibrator (9,15,19).…”

“…fold change). The first is the ratiometric 488/405 measurement detected by the biosensor, the second one is obtained by the parallel analysis of 488 nm/405 nm fluorescence changes of cells expressing the cpVENUS-only control (9,15,19 independent of the biosensor. F0 refers to the ratio of ratios obtained in untreated conditions (F0=1).…”

“…Following this hypothesis, we studied NAD + bioavailability by dissecting the subcellular location of its biosynthesis. To the aim, we exploited a genetically-encoded fluorescent biosensor (9,15,19) to directly monitor free NAD + concentrations in subcellular compartments of MM cells and we connected this information to the biochemical evaluation of subcellular distribution of NAD + biosynthetic enzymes (NBEs). C O N F I D E N T I A L .…”