Amplite® Fluorimetric NAD/NADH Ratio Assay Kit *Red Fluorescence*

1. Prepare 25 µL of NADH standards and/or test samples
2. Add 25 µL of NADH or NAD Extraction Solution
3. Incubate at 37^oC for 15 minutes
4. Add 25 µL of NAD or NADH Extraction Solution
5. Add 75 µL of NAD/NADH working solution
6. Incubate at RT for 15 minutes to 2 hours
7. Monitor fluorescence intensity at Ex/Em = 540/590 nm

It is highly recommended to incubate the cells with Lysis Buffer (Component G) at 37^oC and use the supernatent for the experiment.

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 PBS buffer into the vial of NADH standard (Component C) to have 1 mM (1 nmol/µL) NADH stock solution.

Add 10 mL of NADH Sensor Buffer (Component B) to the bottle of NAD/NADH Recycling Enzyme Mixture (Component A), and mix well.

Note: This NAD/NADH working solution is enough for 125 assays in 96-well plate. The working solution is not stable, use it promptly and avoid direct exposure to light.

Table 1. Layout of NADH standards and test samples in a solid black 96-well microplate. NS = NADH standard (NS1-NS7, 0.03 to 30 µM); BL = blank control; TS = test sample; TS(NADH) = test sample treated with NADH Extraction Solution, then neutralized by NAD Extraction Solution ; TS(NAD) = test sample treated with NAD Extraction Solution , then neutralized by NADH Extraction Solution.

Table 2. Reagent composition for each well. Take note that high concentration of NADH (e.g., >300 µM, final concentration) may cause reduced fluorescence signal due to the over oxidation of NADH sensor (to a non-fluorescent product).

Prepare NADH standards (NS), blank controls (BL), test samples (TS), test samples treated with NADH Extraction Solution (TS(NADH)), and test samples treated with NAD Extraction Solution (TS(NAD)) according to the layout described in Tables 1 and 2.

Note: Prepare cells or tissue samples as desired.

Note: Incubate the cells with Lysis Buffer for 15 mins at 37^oC and use the supernatant for the experiment.

For NAD Extraction (NAD): Add 25 µL of NAD Extraction Solution (Component E) into the wells of NAD/NADH containing test samples. Incubate at 37^oC for 10 to 15 minutes, then add 25 µL of NADH Extraction Solution (Component D) to neutralize the NAD extracts as described in Tables 1 & 2.

For Total NAD and NADH: Add 25 µL of NAD/NADH Control Solution (Component F) into the wells of NADH standards and NAD/NADH containing test samples. Incubate at 37^oC for 10 to 15 minutes, and then add 25 µL of Control Solution (Component F) as described in Tables 1 and 2.

For NADH Extraction (NADH): Add 25 µL of NADH Extraction Solution (Component D) into the wells of NAD/NADH containing test samples. Incubate at 37^oC for 10 to 15 minutes, then add 25 µL of NAD Extraction Solution (Component E) to neutralize the NADH extracts as described in Tables 1 & 2. Note: In healthy mammalian cells, there is more NAD comparing to NADH, so one can simply use total NAD and NADH minus the NAD to calculate the amount of NADH.

1. Add 75 µL of NAD/NADH working solution into each well of NADH standard, blank control, and test samples to make the total NADH assay volume of 150 µL/well.

2. Incubate the reaction at room temperature for 15 minutes to 2 hours (We tested 60 minutes in the figure shown), protected from light.

3. 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. However, the absorption detection has lower sensitivity compared to fluorescence reading.