Amplite® Fluorimetric Hydrogen Peroxide Assay Kit *Red Fluorescence*

Protocol summary

1. Prepare H₂O₂ working solution (50 µL)
2. Add H₂O₂ standards or test samples (50 µL)
3. Incubate at room temperature for 10 - 30 minutes
4. Monitor fluorescence intensity at Ex/Em = 540/590 nm

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

Important notes
The component A is unstable in the presence of thiols such as DTT and β-mercaptoethanol. Thiols higher than 10 uM (final concentration) would significantly decrease the assay dynamic range.

Important notes
NADH and glutathione (reduced form: GSH) may interfere with the assay.

1. Amplite™ Red Peroxidase Substrate stock solution (100X):
Add 250 µL of DMSO (Component E) into the vial of Amplite™ Red Substrate (Component A). This stock solution should be used promptly.

2. Peroxidase stock solution (20 U/mL):
Add 1 mL of Assay Buffer (Component C) into the vial of Horseradish Peroxidase (Component D).

3. H₂O₂ standard solution (20 mM):
Add 22.7 µL of 3% H₂O₂ (0.88 M, Component B) into 977 µL of Assay Buffer (Component C). Note: Diluted H₂O₂ solution is not stable. Any unused portions should be discarded.

Add 50 μL of Amplite™ Red Peroxidase Substrate stock solution (100X) and 200 μL of Peroxidase stock solution (20 U/mL) into 4.75 mL of Assay Buffer (Component C) to make a total volume of 5 mL. Note: Keep from light.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

Table 1. Layout of H₂O₂ standards and test samples in a solid black 96-well microplate. HS = H₂O₂ standard (HS1-HS7, 0.01 to 10 µM); BL = blank control; TS = test sample.

Table 2. Reagent composition for each well. Note that high concentrations of H₂O₂ (e.g., >100 µM, final concentration) may cause reduced fluorescence signals due to the overoxidation of Amplite™ Red (to non-fluorescent products).

H₂O₂ assay in supernatants

1. Prepare H₂O₂ standards (HS), 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 H₂O₂ working solution into each well of H₂O₂ standard, blank control, and test samples to make the total H₂O₂ assay volume of 100 µL/well. For a 384-well plate, add 25 µL of H₂O₂ working solution instead, for a total volume of 50 µL/well.
   
   
3. Incubate the reaction at room temperature for 15 to 30 minutes, protected from light.
   
   
4. Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 540 ± 10 nm, Emission = 590 ± 10 nm (optimal Ex/Em = 540/590 nm). Note: The contents of the plate can also be transferred to a white clear bottom microplate and read by an absorbance microplate reader at the wavelength of 576 ± 5 nm. However, the absorption detection has a lower sensitivity compared to that of a fluorescence reading.
   
   

H₂O₂ assay for cells
The Amplite™ Fluorimetric Hydrogen Peroxide Assay Kit can be used to measure the release of H₂O₂ from cells. The following is a suggested protocol that can be modified to meet the specific research needs.

1. The H₂O₂ cell working solution should be prepared as above, except that the Assay Buffer (Component C) should be replaced with the media that is used in your cell culture system. Suggested medias include (a) Krebs Ringers Phosphate Buffer (KRPB); (b) Hanks Balanced Salt Solution (HBSS); or (c) Serum-free media.
   
   
2. Prepare cells in a 96-well plate (50 - 100 µL/well), and activate the cells as desired. Note: The negative controls (media alone and non-activated cells) are included for measuring background fluorescence. For a 384-well plate, use 25 µL/well of cell media instead.
   
   
3. Add 50 µL of H₂O₂ cell working solution into each well of cells and H₂O₂ standards. For a 384-well plate, add 25 µL of H₂O₂ cell working solution into each well instead.
   
   
4. Incubate the reaction at room temperature for 15 to 30 minutes, protected from light.
   
   
5. Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 540 ± 10 nm, Emission = 590 ± 10 nm (optimal Ex/Em = 540/590 nm).