Amplite® Fluorimetric Myeloperoxidase Assay Kit *Red Fluorescence*

Protocol summary

1. MPO standards or test samples (50 µL)
2. Add MPO 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 E) 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. H₂O₂ stock solution (500X, 10 mM):
Add 10 µL of 3% H₂O₂ (0.88M, Component C) into 870 µL of Assay Buffer (Component B). Note: The diluted H₂O₂ solution is not stable. The unused portion should be discarded.

3. Myeloperoxidase (MPO) standard solution (200 mU/mL):
Add 50 µL of Assay Buffer (Component B) into the vial of Myeloperoxidase Standard (Component D). Note: One vial contains approximately 5 - 10 mU myeloperoxidase.

Add 20 μL of Amplite™ Red Stock Solution (250X) and 10 μL of H₂O₂ (500X) into 5 mL of Assay Buffer (Component B) to make a total volume of 5.03 mL MPO working solution. Protect from light.

Table 1. Layout of MPO standards and test samples in a 96-well solid black microplate. MPO= myeloperoxidase standards (MPO1 - MPO7, 0.01 to 10 mU/mL); BL=blank control; TS = test samples.

Table 2. Reagent composition for each well. Note that high concentration of MPO may cause reduced fluorescence signal due to the over oxidation of Amplite™ Red substrate (to a non-fluorescent product).

1. Prepare myeloperoxidase standards (MPO), 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.
   
   
2. Add 50 µL of MPO working solution to each well of myeloperoxidase standard, blank control, and test samples to make the total MPO assay volume of 100 µL/well. For a 384-well plate, add 25 µL of MPO 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 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm, cut off = 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 that of the fluorescence reading.