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

1. Prepare NADH standards or test samples (50 µL)
2. Add NAD/NADH working solution (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)

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

For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html

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

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.

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.

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).

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.