|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.
Open in Advanced Spectrum Viewer
|Name||Excitation (nm)||Emission (nm)|
|Amplite® ADHP [10-Acetyl-3,7-dihydroxyphenoxazine] *CAS#: 119171-73-2*||571||584|
Authors: Nguyen, Peter Q and Soenksen, Luis R and Donghia, Nina M and Angenent-Mari, Nicolaas M and de Puig, Helena and Huang, Ally and Lee, Rose and Slomovic, Shimyn and Galbersanini, Tommaso and Lansberry, Geoffrey and others,
Journal: Nature Biotechnology (2021): 1366--1374
Authors: Milton, Amber
Authors: Chi, Junjie and Gao, Bingbing and Sun, Mi and Zhang, Fengling and Su, Enben and Liu, Hong and Gu, Zhongze
Journal: Analytical Chemistry (2017)
Authors: Albadawi, Hassan and Chen, John W and Oklu, Rahmi and Wu, Yue and Wojtkiewicz, Gregory and Pulli, Benjamin and Milner, John D and Cambria, Richard P and Watkins, Michael T
Journal: Radiology (2016): 152222
Authors: Pulli, Benjamin and Ali, Muhammad and Iwamoto, Yoshiko and Zeller, Matthias WG and Schob, Stefan and Linnoila, Jenny J and Chen, John W
Journal: Antioxidants & redox signaling (2015): 1255--1269
Authors: Zhang, Yinian and Seeburg, Daniel P and Pulli, Benjamin and Wojtkiewicz, Gregory R and Bure, Lionel and Atkinson, Wendy and Schob, Stefan and Iwamoto, Yoshiko and Ali, Muhammad and Zhang, Wei and others, undefined
Journal: Radiology (2015): 822--830
Authors: Huang, Jiansheng and Smith, Forrest and Panizzi, Peter
Journal: Archives of biochemistry and biophysics (2014): 74--85
Authors: Scherag, Frank D and Br, undefined and stetter, Thomas and Rühe, Jürgen
Journal: Colloids and Surfaces B: Biointerfaces (2014): 576--582
Authors: Pulli, Benjamin and Ali, Muhammad and Forghani, Reza and Schob, Stefan and Hsieh, Kevin LC and Wojtkiewicz, Gregory and Linnoila, Jenny J and Chen, John W
Journal: PLoS One (2013): e67976
Authors: Kozak, Katherine R and Wang, Jianyong and Lye, Melvin and Takkar, Rashi and Kim, Namyong and Lee, Hyunjae and Jeon, Noo Li and Lin, Kedan and Zhang, Crystal and Wong, Wai Lee T and others, undefined
Journal: Lab on a Chip (2013): 1342--1350
Authors: Rajashekhara E, Hosoda A, Sode K, Ikenaga H, Watanabe K.
Journal: Biotechnol Prog (2006): 334
Authors: Fortelius M, Mattjus P.
Journal: Chem Phys Lipids (2006): 103
Authors: Azuine MA, Patel SJ, Lakshman MR.
Journal: Neurochem Int (2006): 67
Authors: Morten KJ, Ackrell BA, Melov S.
Journal: J Biol Chem (2006): 3354
Authors: Mazura P, Fohlerova R, Brzobohaty B, Kiran NS, J and a L., undefined
Journal: J Biochem Biophys Methods (2006): 55
Authors: Rinaldi M, Moroni P, Paape MJ, Bannerman DD.
Journal: Vet Immunol Immunopathol. (2006)
Authors: Fruk L, Muller J, Niemeyer CM.
Journal: Chemistry (2006): 7448
Authors: Drose S, Br and t U, Hanley PJ.
Journal: J Biol Chem (2006): 23733
Authors: Kim GJ, Fiskum GM, Morgan WF.
Journal: Cancer Res (2006): 10377
Authors: Sullivan JC, Sasser JM, Pollock JS.
Journal: Am J Physiol Regul Integr Comp Physiol. (2006)
A Library of Well-Defined and Water-Soluble Poly(alkyl phosphonate)s with Adjustable Hydrolysis
Acetylcholinesterase Inhibitory Activity of Pigment Echinochrome A
Ameliorative Effect of Novel Vitamin Formula with Herbal Extracts on Scopolamine-Induced Alzheimer's Disease
An Increase in Plasma Homovanillic Acid with Cocoa Extract Consumption Is Associated with the Alleviation of Depressive Symptoms in Overweight or Obese Adults
Attenuation of lysyl oxidase and collagen gene expression in keratoconus patient corneal epithelium corresponds to disease severity