Amplite® Fluorimetric Total NADP and NADPH Assay Kit *Red Fluorescence*

Protocol summary

1. Prepare NADP/NADPH working solution (50 µL) 
2. Add NADPH 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. NADPH standard solution (1 mM):
Add 200 µL of PBS buffer into the vial of NADPH Standard (Component C) to make 1 mM (1 nmol/µL) NADPH stock solution. 

Add 10 mL of NADP/NADPH Sensor Buffer (Component B) into the bottle of NADP/NADPH Recycling Enzyme Mixture (Component A) and mix well. Note: This NADP/NADPH working solution is enough for two 96-well plates. The working solution is not stable, use it promptly and avoid direct exposure to light.

Table 1. Layout of NADPH standards and test samples in a solid black 96-well microplate. NS= NADPH Standards (NS1 - NS7, 0.003 to 3 µM), BL=Blank Control, TS=Test Samples.

Table 2. Reagent composition for each well.

1. Prepare NADPH 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. High concentration of NADPH (e.g., >100 µM, final concentration) may cause reduced fluorescence signal due to the over oxidation of NADPH sensor (to a non-fluorescent product).
   
   
2. Add 50 µL of NADPH working solution to each well of NADPH standard, blank control, and test samples to make the total NADPH assay volume of 100 µL/well. For a 384-well plate, add 25 µL of NADPH 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, Emission = 590 - 600 nm (optimal at 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 NADP/NADPH ratio measurements, Cat No. 15264 is recommended. For cell based NADP/NADPH measurements, ReadiUse™ mammalian cell lysis buffer *5X* (Cat No. 20012) is recommended to use for lysing the cells.