Amplite® IR

Protocol summary

1. Prepare 100 µM Amplite^TM IR with 0.8 U/mL peroxidase in phosphate buffer and add 50 µL in a well
2. Add H₂O₂ standards or test samples (50 µL)
3. Incubate at RT for 0-30 minutes
4. Monitor fluorescence intensity at Ex/Em = 640/680 nm

Important notes
The following is the recommended protocol for H₂O₂ assay in solution and live cells. The protocol only provides a guideline, should be modified according to the specific needs.

Amplite^TM IR stock solution:
Add appropriate amount of anhydrous DMSO to make 10 to 25 mM Amplite^TM IR stock solution.

Amplite^TM IR working solution(2X):
In order to achieve final concentration per well of 50 to 100 µM in 50 mM phosphate buffer or buffer of your choice, make 100 to 200 µM concentration solution in a tube. 50 µL is required per well. Note: Amplite^TM IR is unstable in the presence of thiols such as DTT and b-mercaptoethanol. Thiols higher than 10 μM (final concentration) could significantly decrease the assay dynamic range. NADH and glutathione (reduced from: GSH) may interfere with the assay. Note: We recommend using fresh stock solution every time you perform experiments.

Run H₂O₂ assay in supernatants

1. Add 50 µL of 2X Amplite^TM IR working solution (from Step 1.2) into each well of the H₂O₂ standard, blank control, and test samples to make the total H₂O₂ assay volume of 100 µL/well. Note: For a 384-well plate, add 25 µL of sample and 25 µL of 2X Amplite^TM IR working solution into each well.
   
   
2. Incubate the reaction at room temperature for 0 to 30 minutes, protected from light.
   
   
3. Monitor the fluorescence increase at Ex/Em = 640/680 nm with a fluorescence plate reader. Note: Amplite™ IR peroxidase substrate is easy to be self-oxidized, so read the fluorescence as soon as the H₂O₂ reaction mixture is added to increase the signal to noise ratio.
   
   
4. The fluorescence in blank wells (with the assay buffer only) is used as a control, and is subtracted from the values for those wells with the H₂O₂

 Run H₂O₂ assay for cells:

1. Amplite™ IR can be used to measure the release of H₂O₂ from cells. The following is a suggested protocol that can be modified for your specific research needs.The Amplite^TM IR working solution should be prepared as Step 1.2 except that the phosphate buffer should be replaced with the media that is used in the 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 background fluorescence.
   
   
3. Add 50 µL of H₂O₂ reaction mixture to each well of the cells, and those of H₂O_2.Note: For a 384-well plate, add 25 µL of cells and 25 µL of H₂O₂ reaction mixture into each well.
   
   
4. Incubate the reaction for 0 to 30 minutes at room temperature, protected from light.
   
   
5. Monitor the fluorescence increase at Ex/Em = 640/ 680 nm with a fluorescence plate reader. 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 670 nm. The absorption detection has lower sensitivity compared to fluorescence reading. Note: The fluorescence background increases with time, thus it is important to subtract the fluorescence intensity value of the blank wells for each data point.