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Amplite® Fluorimetric Total NAD and NADH Assay Kit *Red Fluorescence*

NADH dose response was measured with Amplite® Total NAD and NADH Assay Kit in a solid black 96-well plate using a NOVOStar microplate reader (BMG Labtech). RFU at Ex/Em = 540/590 nm.
NADH dose response was measured with Amplite® Total NAD and NADH Assay Kit in a solid black 96-well plate using a NOVOStar microplate reader (BMG Labtech). RFU at Ex/Em = 540/590 nm.
NADH dose response was measured with Amplite® Total NAD and NADH Assay Kit in a solid black 96-well plate using a NOVOStar microplate reader (BMG Labtech). RFU at Ex/Em = 540/590 nm.
Effects of AA005 on ATP production and AMPK/mTOR signaling pathway. (A) Chemical structure of AA005-fluorescein. (B) The intracellular localization of AA005. The cells were co-incubated with AA005-flu at 100 nM for 12 h, and analyzed by confocal microscopy using a mitotracker (red) to counter-stain mitochondria. (C) MTT assay of LOVO, HT29, HCT116 and HBEpiC cells upon AA005-flu at indicated concentrations for 48 h. (D) AA005 decreases the mitochondrial transmembrane potential of colon cancer cells revealed by increase in Rhodamine 123-negative cells. The cells were treated with AA005 at indicated concentration for 24 h and analyzed by Rhodamine 123 staining and flow cytometry. (E) The cells were treated with or without AA005 at indicated concentration for 24 h, NAD+/NADH ratio was measured using an Amplite<sup>TM</sup> Colorimetric NAD/NADH Assay Kit. (F) The cells were treated with or without AA005 at indicated concentration for 24 h, and ATP content was measured using an ATP Bioluminescence Assay Kit. (G) The cells were treated with AA005 at indicated concentration and time points, lysed, and Western blot analysis was performed using indicated antibodies. Source: Graph from <strong>Identification of an Annonaceous Acetogenin Mimetic, AA005, as an AMPK Activator and Autophagy Inducer in Colon Cancer Cells</strong> by Yong-Qiang Liu et al., <em>PLOS</em>, Oct. 2012.
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H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


Nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) are two important cofactors found in cells. NADH is the reduced form of NAD+, and NAD+ is the oxidized form of NADH. It forms NADP with the addition of a phosphate group to the 2' position of the adenyl nucleotide through an ester linkage. NADP is used in anabolic biological reactions, such as fatty acid and nucleic acid synthesis, which require NADPH as a reducing agent. In chloroplasts, NADP is an oxidizing agent important in the preliminary reactions of photosynthesis. The NADPH produced by photosynthesis is then used as reducing power for the biosynthetic reactions in the Calvin cycle of photosynthesis. The traditional NAD/NADH and NADP/NADPH assays are done by monitoring of NADH or NADPH absorption at 340 nm. This method suffers low sensitivity and high interference since the assay is done in the UV range that requires expensive quartz microplate. This Amplite® NAD/NADH Assay Kit provides a convenient method for sensitive detection of NAD and NADH. The enzymes in the system specifically recognize NAD/NADH in an enzyme cycling reaction. There is no need to purify NAD/NADH from sample mix. The enzyme cycling reaction significantly increases detection sensitivity. In addition, this assay has very low background since it is run in the red visible range that significantly reduces the interference from biological samples. The assay has demonstrated high sensitivity and low interference with 570 nm excitation 590 nm emission.


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black


Example protocol


Protocol summary

  1. Prepare NAD/NADH working solution (50 µL)
  2. Add NADH standards or test samples (50 µL)
  3. Incubate at room temperature for 15 minutes – 2 hours
  4. Monitor the fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 570 nm)

Important notes
Thaw one of each kit component at room temperature before starting the experiment.


Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. NADH standard solution (1 mM):
Add 200 µL of 1X PBS buffer into the vial of NADH Standard (Component C) to make 1 mM (1 nmol/µL) NADH standard solution.


NADH standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/15257

Add 10 µL of 1 mM (1 nmol/µL) NADH standard solution to 990 µL 1X PBS buffer to generate 10 µM (10 pmol/µL) NADH standard solution (NS7). Take 10 µM NADH standard solution (NS7) to perform 1:3 serial dilutions in 1X PBS buffer to get serially diluted NADH standards (NS6 - NS1). Note: Diluted NADH standard solution is unstable and should be used within 4 hours.


Add 10 mL of NADH Sensor Buffer (Component B) into the bottle of NAD/NADH Recycling Enzyme Mix (Component A) and mix well to make NAD/NADH working solution. Note: This NAD/NADH working solution is enough for two 96-well plates.

For guidelines on cell sample preparation, please visit


Table 1. Layout of NADH standards and test samples in a solid black bottom 96-well microplate. NS=NADH Standards (NS1 - NS7, 0.014 to 10 µM) , BL=Blank Control, TS=Test Samples.


Table 2. Reagent composition for each well. High concentration of NADH (e.g., >100 µM, final concentration) may cause reduced fluorescence signal due to the over oxidation of NADH sensor (to a non-fluorescent product).

NS1 - NS750 µLSerial Dilutions (0.014 to 10 µM)
BL50 µL1X PBS buffer
TS50 µLtest sample
  1. Prepare NADH standards (NS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL. Note: Prepare cells or tissue samples as desired.

  2. Add 50 µL of NAD/NADH working solution to each well of NADH standard, blank control, and test samples to make the total NAD/NADH assay volume of 100 µL/well. For a 384-well plate, add 25 µL of NAD/NADH working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate the reaction at room temperature for 15 minutes to 2 hours, protected from light.

  4. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm). Note:The contents of the plate can also be transferred to a white clear bottom plate and read by an absorbance microplate reader at the wavelength of 576 ± 5 nm. The absorption detection has lower sensitivity compared to fluorescence reading. Note: For NAD/NADH ratio measurements, kit 15263 is recommended. Note: For cell based NAD/NADH measurements, ReadiUse™ mammalian cell lysis buffer *5X* (cat#20012) is recommended to use for lysing the cells.



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