Amplite® Fluorimetric Xanthine Oxidase Assay Kit *Red Fluorescence*

Protocol summary

1. XO standards or test samples (50 µL)
2. Add XO working solution (50 µL)
3. Incubate at room temperature for 15 - 30 minutes
4. Read fluorescence intensity at Ex/Em = 540/590 nm (cut off 570 nm)

Important notes
Thaw all the kit components to room temperature before starting the experiment.

1. Amplite™ Red stock solution (250X):
Add 40 µL of DMSO (Component F) into the vial of Amplite™ Red substrate (Component A). The stock solution should be used promptly. Note: The Amplite™ Red substrate is unstable in the presence of thiols such as dithiothreitol (DTT) and 2-mercaptoethanol. The final concentration of DTT or 2-mercaptoethanol in the reaction should be no higher than 10 µM. The Amplite™ Red substrate is also unstable at high pH (>8.5). Therefore, the reaction should be performed at pH = 7 – 8. The provided assay buffer, pH = 7.4, is recommended.

2. HRP stock solution (500X):
Add 100 µL of Assay Buffer (Component B) into the vial of Horseradish Peroxidase (Component C).

3. Xanthine Oxidase (XO) standard solution (1 U/mL)
Add 200 µL of Assay Buffer (Component B) into the vial of Xanthine Oxidase Standard (Component E).

Add 20 μL of Amplite™ Red stock solution (250X), 10 μL of HRP stock solution (500X), and 50 μL of Xanthine (100X, Component D) into 5 mL of Assay Buffer (Component B) to make a total volume of 5.08 mL Xanthine Oxidase (XO) working solution. Protect from light.

Table 1. Layout of XO standards and test samples in a solid black 96-well microplate.

Table 2. Reagent composition for each well.

1. Prepare XO standards (XO), blank controls (BL), and test samples (TS) into a solid black 96-well microplate 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 XO working solution into each well of the XO standards, blank control, and test samples to make the total XO assay volume of 100 µL/well. For a 384-well plate, add 25 µL of XO working solution into each well instead, for a total volume of 50 µL/well.
   
   
3. Incubate the reaction for 15 to 30 minutes at room temperature, protected from light.
   
   
4. Monitor the fluorescence intensity with a fluorescence plate reader at Excitation = 530 - 570 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm), cut off = 570 nm.