Amplite® Fluorimetric NADH Assay Kit *Red Fluorescence*

Protocol summary

1. Prepare 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 fluorescence intensity at Ex/Em = 540/590 nm

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

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

Add 10 mL of Amplite™ NADH Assay Buffer (Component B) to the bottle of NADH Recycling Enzyme Mixture (Component A) and mix well. Note: This NADH working solution is enough for two 96-well or four 384-well plates. 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, 0.1 to 100 µM); BL = blank control; TS = test sample.

Table 2. Reagent composition for each well.

1. Prepare NADH standards (NS), blank controls (BL), and test samples (TS) according to the layout provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
   
   
2. Add 50 µL of NADH working solution into each well of NADH standard, blank control, and test samples to make the total NADH assay volume of 100 µL/well. For a 384-well plate, add 25 µL of 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 Excitation = 530 - 570 nm, Emission = 590 - 600 nm (optimal 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 will have a lower sensitivity compared to that of the fluorescence reading. For cell based NADH measurements, ReadiUse™ mammalian cell lysis buffer *5X* (Cat No. 20012) is recommended to use for lysing the cells.