Amplite® Fluorimetric Monoamine Oxidase Assay Kit *Red Fluorescence*

Protocol summary

1. MAO standards or test samples (50 µL)
2.  Add MAO working solution (50 µL)
3. Incubate at room temperature for 30-60 min 
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 (200X):
Add 100 µL of Assay Buffer (Component B) into the vial of horseradish peroxidase (Component C).

3. Plasma Amine Oxidase (PAO) standard solution (20 U/mL):
Add 125 µL of Assay Buffer (Component B) into the vial of Plasma Amine Oxidase Standard (Component E).

Add 20 μL of Amplite™ Red stock solution (250X), 25 µL of HRP stock solution (200X) and 25 µL of MAO Substrate (Component D) into 5 mL of Assay Buffer (Component B) to make a total volume of 5.07 mL Monoamine Oxidase (MAO) working solution. Protect from light.

Table 1. Layout of PAO standards and test samples in a solid black 96-well microplate. PAO = plasma amine oxidase standard (PAO1 - PAO7, 1 to 1000 mU/mL); BL = blank control; TS = test sample.

Table 2. Reagent composition for each well

1. Prepare plasma amine oxidase standards (PAO), blank controls (BL), and test samples (TS) into a 96-well solid black microplate according the 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 MAO working solution into each well of the PAO standard, blank control, and test samples to make the total PAO assay volume of 100 µL/well. For a 384-well plate, add 25 µL of MAO working solution into each well instead, for a total volume of 50 µL/well.
   
   
3. Incubate the reaction for 30 to 60 minutes at room temperature, protected from light.
   
   
4. Monitor the fluorescence intensity with a fluorescence plate reader at Excitation = 530 - 570, Emission = 590 - 600 nm (optimal 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.