Amplite® Fluorimetric NADP/NADPH Ratio Assay Kit *Red Fluorescence*

1. Prepare 25 µL NADPH standards and/or test samples
2. Add 25 µL of NADPH or NADP Extraction Solution
3. Incubate at 37^oC for 15 minutes
4. Add 25 µL of NADP or NADPH Extraction Solution
5. Add 75 µL NADP/NADPH working solution

6. Incubate at 37 °C for 15 minutes – 2 hours

7. Monitor fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 570 nm)

It is highly recommended to incubate the cells with Lysis Buffer (Component G) at 37^oC and use the supernatant 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 NADPH standard (Component C) to make 1 mM (1 nmol/µL) NADPH standard solution.

Add 10 mL of NADPH Sensor Buffer (Component B) into the bottle of NADP/NADPH Recycling Enzyme Mixture (Component A) and mix well to make NADP/NADPH working solution.
Note        This NADP/NADPH working solution is enough for 125 assays in 96-well plate.

Table 1. Layout of NADPH standards and test samples in a solid black 96-well microplate. NS= NADPH Standards (NS1 - NS7, 0.01 to 10 µM); BL=Blank Control; TS=Test Samples; TS (NADPH) = Test Samples treated with NADPH Extraction Solution, then neutralized by NADP Extraction Solution; TS (NADP) = Test Samples treated with NADP Extraction Solution, then neutralized by NADPH Extraction Solution.

Table 2. Reagent composition for each well. Note: 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).

1. Prepare NADPH standards (NS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2.
   
   Note        Prepare cells or tissue samples as desired. NADP/NADPH Lysis Buffer (Component G) can be used for lysing the cells for convenience. One can simply use total NADP and NADPH minus the NADP to calculate the amount of NADPH.
   
   Note        It is highly recommended to incubate the cells at 37 °C and use the supernatant for the experiment.
2. For NADP Extraction (NADP): Add 25 µL of NADP Extraction Solution (Component E) into the wells of NADP/NADPH containing test samples. Incubate at 37 °C for 10 to 15 minutes, then add 25 µL of NADPH Extraction Solution (Component D) to neutralize the NADP extracts as described in Tables 1 & 2.For Total NADP and NADPH: Add 25 µL of NADP/NADPH Control Solution (Component F) into the wells of NADPH standards and NADP/NADPH containing test samples. Incubate at 37 °C for 10 to 15 minutes, and then add 25 µL of Control Solution (Component F) as described in Tables 1 & 2.For NADPH Extraction (NADPH): Add 25 µL of NADPH Extraction Solution (Component D) into the wells of NADP/NADPH containing test samples. Incubate at 37 °C for 10 to 15 minutes, then add 25 µL of NADP Extraction Solution (Component E) to neutralize the NADPH extracts as described in Tables 1 & 2.
3. Add 75 µL of NADPH working solution into each well of NADPH standard, blank control, and test samples to make the total NADPH assay volume of 150 µL/well.
4. Incubate the reaction at 37 °C for 15 minutes to 2 hours (We tested 60 minutes in the figure shown), protected from light.
   
   Note        In some cases, incubation time can be increased to more than 2 hours.
5. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 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.