Amplite® Fluorimetric Hydrogen Peroxide Assay Kit *Near Infrared 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 0 - 30 minutes
4. Monitor fluorescence intensity at Ex/Em = 640/680 nm (Cutoff = 665 nm)

Important notes
Thaw all the kit components at room temperature before starting the experiment. 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. 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) to make 100X Amplite^TM IR Peroxidase Substrate stock solution. Note: 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 µM, it would significantly decrease the assay dynamic range.

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

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) to make 20 mM H₂O₂ standard solution. Note: The diluted H₂O₂ stock solution is not stable. The unused portion should be discarded.

Add 50 μL of 100X Amplite™ IR Peroxidase Substrate stock solution and 200 μL of 20 U/mL Peroxidase stock solution into 4.75 mL of Assay Buffer (Component C) to make H₂O₂ working solution. Keep from light.

Table 1. Layout of H₂O₂ standards and test samples in a solid black 96-well microplate. HS= H₂O₂ Standards (HS1 - HS7, 0.01 to 10 µM); BL=Blank Control; TS=Test Samples

Table 2. Reagent composition for each well.

Run 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 0 to 30 minutes, protected from light.
   
   
4. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 640/680 nm (Cutoff =665nm). Note: Amplite™ IR Peroxidase Substrate is easy to be self-oxidized, so read the fluorescence as soon as the H₂O₂ working solution was added to increase the signal to noise ratio. 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 650 nm. The absorption detection has lower sensitivity compared to the fluorescence reading.

Run H₂O₂ assay for cells:

1. The H₂O₂ working solution should be prepared as above 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 working solution into each well of cells and H₂O₂ standards. For a 384-well plate, add 25 µL of cells and 25 µL of H₂O₂ working solution into each well.
   
   
4. Incubate the reaction at room temperature for 0 to 30 minutes, protected from light.
   
   
5. Monitor the fluorescence intensity with a fluorescence plate reader at Ex/Em = 640/680 nm (Cutoff = 665nm).