Feb 03, 2025

Public workspaceNAD consumption assay

DOI
dx.doi.org/10.17504/protocols.io.5jyl8d5y9g2w/v1
  • 1Institut Imagine
  • Team Deleidi
Federico Bertoli
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DOI: dx.doi.org/10.17504/protocols.io.5jyl8d5y9g2w/v1
Protocol Citation: Maria Jose Perez J. 2025. NAD consumption assay. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8d5y9g2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 26, 2024
Last Modified: February 03, 2025
Protocol Integer ID: 112828
Funders Acknowledgements:
ASAP
Abstract
NAD consumption assay
NAD consumption assay
NAD consumption assay
Detach 1 × 10^5 iPSC-derived microglia per condition using Accutase.
Centrifuge the cells and resuspend them in 100 µL of PBS per well.
Add 80 µM of the fluorescent NAD analog nicotinamide 1,N6-ethenoadenine dinucleotide (Sigma‒Aldrich) to each well.
Excite the fluorescent NAD analog at 340 nm and measure fluorescence at 460 nm.
Perform fluorescence measurements at 37°C every minute for 1 hour using a SpectraMax M2 Multi-Mode microplate reader (Molecular Devices).
Calculate NADase activity as the slope of the linear portion of the fluorescence-time curve.
Normalize NADase activity to the protein concentration of each sample.
Determine protein concentrations using the Pierce‱ BCA Protein Assay Kit (Cat. number 23225, Thermo Fisher Scientific).