Amplite® Colorimetric Hydrogen Peroxide Assay Kit

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 - 60 minutes
4. Monitor absorbance at 650 nm

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

Important notes
Amplite™ IR Peroxidase Substrate (Component A) is unstable in the presence of thiols such as DTT and β-mercaptoethanol. If the final concentration of the thiols is higher than 10 uM, it would significantly decrease the assay dynamic range. 

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

1. Amplite™ IR Peroxidase Substrate stock solution (100X):
Add 250 µL of DMSO (Component E) into the vial of Amplite™ IR Peroxidase Substrate (Component A).

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: The diluted H₂O₂ stock solution is not stable. The unused portion should be discarded.

Add 50 μL of Amplite™ IR 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 white wall/clear bottom 96-well microplate. HS= H₂O₂ Standards (HS1 - HS7, 1.563 to 100 µM); BL=Blank Control; TS=Test Samples.

Table 2. Reagent composition for each well.

H2O2 assay in supernatants reaction

1. Prepare H₂O₂ standards (HS), 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 H₂O₂ working solution to 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 into each well instead, for a total volume of 50 µL/well.
   
   
3. Incubate the reaction at room temperature for 10 to 30 minutes, protected from light.
   
   
4. Monitor the absorbance with an absorbance plate reader at 650 nm.
   
   

H2O2 assay for cells
The Amplite™ Colorimetric 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 given except that the Assay Buffer (Component C) should be replaced with the media used in your cell culture system. Suggested media including (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 the background fluorescence.
   
   
3. Add 50 µL of H₂O₂ cell working solution to each well of cells and H₂O₂ standards to make the total H₂O₂ assay volume of 100 µL/well. For a 384-well plate, add 25 µL of H₂O₂ cell working solution into each well instead, for a total volume of 50 µL/well.
   
   
4. Incubate the reaction at room temperature for 10 to 60 minutes, protected from light.
   
   
5. Monitor the absorbance with an absorbance plate reader at 650 nm.