EnzyFluo™ NAD/NADH Assay Kit

-20 °C

Can I determine NAD/NADH directly in 96-well plates?
Yes, NAD/NADH can be determined directly in 96-well plate. Please see our optimized protocol Direct NAD/NADH Assays in 96-Well Plate

When tissue is used, should it be freshly obtained? Or is -80C storage ok?
If direct sample processing is not possible, we recommend snap freezing tissue samples in liquid nitrogen and keeping them either in liquid nitrogen or at -80C until further processing.

I have conducted protocols that involve trypsinizing cells, washing with PBS, pelleting, and then applying extraction buffer. My concern with this method is that PBS is known to cause mitochondrial fragmentation very soon after application to cells. Obviously, the functionality of the mitochondria is very important for NADH/NAD levels. I developed an alternative by keeping my cells adhered to the plate, washing once with pbs, applying heated extraction buffer to the cells directly and then scraping cells off. Do you have any thoughts or experience that would indicate whether one method is better than the other?
The second method is better because the first method may cause mitochondrial fragmentation and thus may alter intracellular NAD/NADH concentrations. The second method involves less pretreatment. Adding the heated extraction buffer terminates the metabolic reactions in the cells and is thus much less likely to alter the intracellular NAD/NADH concentrations.

I have also done many experiments with different numbers of cells. What I notice is that there seems to be a sigmoidal relationship between the number of cells and the NAD and NADH concentrations. I am using C2C12 and H9C2 cells. Do you have any experience with these cells at different seeding densities? Also, do you consistently see a relationship between number of cells NAD(H) concentrations? Is this relationship linear, exponential, sigmoidal?
We have not done these experiments. Your results are very interesting, and not unexpected. In general, cells are healthy at certain densities. When cells lack contacts for a healthy environment at low density, they are likely to produce less NAD/NADH, and if too dense, the cells become less active and thus would reach a plateau in NAD/NADH concentrations.


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