Amplite® Fluorimetric Hydrogen Peroxide Assay Kit *Red Fluorescence*
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
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Shipping | Standard overnight for United States, inquire for international |
Excitation (nm) | 571 |
Emission (nm) | 584 |
Certificate of Origin | Download PDF |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12171501 |
Overview | SDSProtocol |
Excitation (nm) 571 | Emission (nm) 584 |
Platform
Fluorescence microplate reader
Excitation | 540 nm |
Emission | 590 nm |
Cutoff | 570 nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
Protocol summary
- Prepare H2O2 working solution (50 µL)
- Add H2O2 standards or test samples (50 µL)
- Incubate at room temperature for 10 - 30 minutes
- 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.
PREPARATION OF STOCK SOLUTION
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.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11501
Add 1 µL of 20 mM H2O2 stock solution into 1999 µL of Assay Buffer (Component C) to get a 10 µM H2O2 standard (HS7). Take 10 µM H2O2 standard and perform 1:3 serial dilutions to get serial dilutions of H2O2 standard (HS6 - HS1).
PREPARATION OF WORKING SOLUTION
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
SAMPLE EXPERIMENTAL PROTOCOL
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.
BL | BL | TS | TS |
HS1 | HS1 | ... | ... |
HS2 | HS2 | ... | ... |
HS3 | HS3 | ||
HS4 | HS4 | ||
HS5 | HS5 | ||
HS6 | HS6 | ||
HS7 | HS7 |
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).
Well | Volume | Reagent |
HS1 - HS7 | 50 µL | serial dilution (0.01 to 10 µM) |
BL | 50 µL | Assay Buffer (Component C) |
TS | 50 µL | sample |
H2O2 assay in supernatants
- 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.
- 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.
- Incubate the reaction at room temperature for 15 to 30 minutes, protected from light.
- 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.
- 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.
- 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.
- 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.
- Incubate the reaction at room temperature for 15 to 30 minutes, protected from light.
- Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 540 ± 10 nm, Emission = 590 ± 10 nm (optimal Ex/Em = 540/590 nm).
Product Family
Name | Excitation (nm) | Emission (nm) |
Amplite® Fluorimetric Hydrogen Peroxide Assay Kit *Near Infrared Fluorescence* | 648 | 668 |
Images
Total RNA was isolated from these cultures and subjected to qRT-PCR analysis for PPARα (A). The expression of the house keeping gene HPRT was used for normalization. Values ± SEM represent the mean relative fold induction from three independent experiments. **P ≤0.01; ***P ≤0.001. Dual luciferase reporter activity of PPRE was measured in C22 cells treated either with control (Con) or GW9662 (B). The activity of luciferase was measured in cell lysates and normalized to the activity of renilla. (E.V-empty vector). Data represent ± SD of three independent experiments, P value, unpaired Student t-test. The culture supernatants were collected subjected to H2O2 assay as per manufacture instructions (C).Source: PPARα-mediated peroxisome induction compensates PPARγ-deficiency in bronchiolar club cells by Srikanth Karnati et al., PLOS, Sept 2018.
Citations
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Journal: International Journal of Nanomedicine (2023): 6257--6274
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Application notes
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