PrincipleNAD/NADH Assay Kit-WST enables quantification of the amount of total NAD+/NADH, NADH and NAD+ in cells and measurement of their ratio.

Measurement of NAD⁺ and NADH

Cell lysate from a cell culture can be easily prepared via Deproteinzation using the extraction buffer and filtration tubes found within this kit. Intracellular NADH levels can be quantified by heat treatment of cell lysate. Additionally, intracellular NAD⁺levels can be determined by subtracting NADH levels from independently measured total NAD⁺/NADH levels.

Study of NAD⁺/NADH as MarkersRecently, it has become clear that Sirtuin is linked to longevity and plays a role in NAD+ level regulation. Also Sirtuin has been recognized as a marker necessary to understanding biological states, such as obesity & diabetes, as well as cellular differentiation.

In the experiment shown below, the NAD⁺ /NADH levels & ratio were determined using HeLa cells.

Standard curves were constructed using different concentrations of HeLa cells (2.5×10⁵ and 5.0×10⁵ cells) cultured in growth media. The standard curves were then used to determine the intracellular NAD⁺ and NADH levels. As a result, NAD⁺ and NADH levels varied depending on cell number while the change in cell number had no effect on the NAD⁺/NADH ratio.

Measurement of NAD⁺/NADH in Combination with Lactate Assay KitChange in metabolic activity was observed when the glycolytic inhibitor 2-Deoxy-D-glucose was added to HeLa cells.

2-Deoxy-D-glucose was added to HeLa cells (1×10⁶ cells) to obtain a final concentration of 6 mmol/l 2-Deoxy-D-glucose. After 24 hours of incubation, lactate levels in the supernatant were quantified using the Lactate Assay Kit-WST (Item#: L256), and the NAD⁺/NADH ratio was determined with the cell pellet after removing the supernatant using the NAD/NADH Assay Kit-WST.

As a result, intracellular glycolysis was inhibited by 2-Deoxy-D-glucose, which led to decreased lactate levels and an increase in the NAD⁺/NADH ratio.

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