|Shipping||Standard overnight for United States, inquire for international|
|H-phrase||H303, H313, H333|
|Intended use||Research Use Only (RUO)|
|R-phrase||R20, R21, R22|
|Storage||Freeze (< -15 °C); Desiccated; Minimize light exposure|
Fluorescence microplate reader
|Recommended plate||Solid black|
AT A GLANCE
- Prepare 100 µM AmpliteTM IR with 0.8 U/mL peroxidase in phosphate buffer and add 50 µL in a well
- Add H2O2 standards or test samples (50 µL)
- Incubate at RT for 0-30 minutes
- Monitor fluorescence intensity at Ex/Em = 640/680 nm
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.
PREPARATION OF STOCK SOLUTION
AmpliteTM IR stock solution:
Add appropriate amount of anhydrous DMSO to make 10 to 25 mM AmpliteTM IR stock solution.
PREPARATION OF WORKING SOLUTION
AmpliteTM 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: AmpliteTM 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.
SAMPLE EXPERIMENTAL PROTOCOL
Run H2O2 assay in supernatants
- Add 50 µL of 2X AmpliteTM 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 AmpliteTM IR working solution into each well.
- Incubate the reaction at room temperature for 0 to 30 minutes, protected from light.
- 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.
- 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:
- 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 AmpliteTM 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.
- 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.
- Add 50 µL of H2O2 reaction mixture to each well of the cells, and those of H2O2.Note: For a 384-well plate, add 25 µL of cells and 25 µL of H2O2 reaction mixture into each well.
- Incubate the reaction for 0 to 30 minutes at room temperature, protected from light.
- 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.
Common stock solution preparation
|0.1 mg||0.5 mg||1 mg||5 mg||10 mg|
|1 mM||250 µL||1.25 mL||2.5 mL||12.5 mL||25 mL|
|5 mM||50 µL||250 µL||500 µL||2.5 mL||5 mL|
|10 mM||25 µL||125 µL||250 µL||1.25 mL||2.5 mL|
|Mass (Calculate)||Molecular weight||Volume (Calculate)||Concentration (Calculate)||Moles|
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