Cell Meter™ Mitochondrial Hydroxyl Radical Detection Kit *Red Fluorescence*
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Catalog Number | |
Unit Size | |
Quantity |
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
Excitation (nm) | 576 |
Emission (nm) | 598 |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Overview | ![]() ![]() |
Excitation (nm) 576 | Emission (nm) 598 |
Platform
Fluorescence microscope
Excitation | Cy3/TRITC filter set |
Emission | Cy3/TRITC filter set |
Recommended plate | Black wall/clear bottom |
Fluorescence microplate reader
Excitation | 540 nm |
Emission | 590 nm |
Cutoff | 570 nm |
Recommended plate | Black wall/clear bottom |
Instrument specification(s) | Bottom read mode |
Components
Example protocol
AT A GLANCE
Protocol summary
- Prepare cells
- Incubate cells with MitoROS™ OH580 working solution at 37°C for 60 minutes
- Incubate cells with test compounds (to induce OH-)
- Monitor the fluorescence increase at Ex/Em= 540/590 nm
Important notes
Thaw all the components at room temperature before use.
PREPARATION OF STOCK SOLUTION
1. MitoROS™ OH580 stock solution (500X):
Add 50 µL of DMSO (Component C) into the vial of MitoROS™ OH580 (Component A), and mix them well. Note: 25 uL of stock solution is enough for 1 plate. Note: Unused portion can be aliquoted and stored at ≤ -20ºC for more than one month if the tubes are sealed tightly and kept from light. Avoid repeated freeze-thaw cycles.
PREPARATION OF WORKING SOLUTION
Add 25 μL of 500X DMSO reconstituted MitoROS™ OH580 stock solution into 10 mL of Assay Buffer (Component B). Mix well. Note: This working solution is stable for at least 2 hours at room temperature.
For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html
SAMPLE EXPERIMENTAL PROTOCOL
- Remove medium, and add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of MitoROS™ OH580 working solution into the cell plate. Incubate cells at 37°C for 60 minutes.
- To induce hydroxyl radical, treat cells with test compounds in your desired buffer (such as PBS or HHBS) at 37°C for a desired period of time, protected from light. Note: We treated HeLa cells with Fenton reaction (10 µM CuCl2 and 100 µM H2O2) at 37°C for 1 hour to induce exogenous hydroxyl radical. See Figure 1 for details. We treated RAW 264.7 cells with PMA (phorbol 12-myristate 13-acetate) in growth medium at 37°C for 4 hours to stimulate endogenous hydroxyl radical.
- Wash cells 2 - 3 times with HHBS or DPBS, and add 100 µL Assay Buffer (Component B) to each well.
- Monitor the fluorescence signal in cells using fluorescence microscope with a TRITC filter set, or measure fluorescence increase using fluorescence microplate reader at Ex/Em = 540/590 nm (cut off = 570 nm) with bottom read mode.
Images



Citations
Authors: Asanuma, Kanna and Yokota, Seiji and Chosa, Naoyuki and Kamo, Masaharu and Ibi, Miho and Mayama, Hisayo and Iri{\'e}, Tarou and Satoh, Kazuro and Ishisaki, Akira
Journal: Journal of Oral Biosciences (2022)
Authors: Zhang, Xuyang and Liu, Zhi and Yang, Wenqin and Zhao, Fengchun and Zhang, Chao and Feng, Hui and Zhou, Tengyuan and Zhong, Jun and Zou, Yongjie and Feng, Hua and others,
Journal: Oxidative Medicine and Cellular Longevity (2022)
Authors: Itoh, Johbu and Itoh, Yoshiko
Journal: Frontiers in Bioscience-Scholar (2022): 27
Authors: Nie, Hongyun and Nie, Maiqian and Diwu, Zhenjun and Wang, Lei and Qiao, Qi and Zhang, Bo and Yang, Xuefu
Journal: Environmental Research (2020): 110159
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