Cell Meter™ Mitochondrial Hydroxyl Radical Detection Kit *Red Fluorescence*
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Unit Size | |
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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
![Fluorescence images of hydroxyl radical measurement in HeLa cells using MitoROS™ OH580 (Cat#16055). HeLa cells were incubated with MitoROS™ OH580 working solution at 37 °C for 1 hour, then washed once with HHBS. Fenton Reaction: Cells were then treated with 10 µM CuCl2 and 100 µM H<sub>2</sub>O<sub>2</sub> in 1X HBSS buffer at 37 °C for 1 hour. Control: HeLa cells were kept in 1X HBSS buffer without treatment. After washing 3 times with HHBS, HeLa cells were measured using a fluorescence microscope with a TRITC filter set (Red). Cell nuclei were stained with Hoechst 33342 (Cat#17530, Blue).](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcell-meter-mitochondrial-hydroxyl-radical-detection-kit-red-fluorescence%2Ffigure-for-cell-meter-mitochondrial-hydroxyl-radical-detection-kit-red-fluorescence_ktAAE.jpg&w=3840&q=75)
![Detection of intracellular hydroxyl radical in RAW 264.7 cells using MitoROS™OH580 (Cat#16055). Cells were incubated with MitoROS™ OH580 working solution at 37 ºC for 1 hour, then washed once with HHBS. Cells were then incubated without or with PMA (phorbol 12-myristate 13-acetate, 10 to 500 ng/mL) in growth medium at 37ºC for 4 hours. After washing 3 times with HHBS, HeLa cells were measured using a fluorescence microscope with a TRITC filter set.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcell-meter-mitochondrial-hydroxyl-radical-detection-kit-red-fluorescence%2Ffigure-for-cell-meter-mitochondrial-hydroxyl-radical-detection-kit-red-fluorescence_uCaxU.jpg&w=3840&q=75)
![Antioxidant NAC partially and significantly reverses the H2O2-mediated increase in phosphorylation of ERK1/2 in FLS1 cells. (A) Cells were cultured (Aa) without or (Ab) with H2O2 (500 μM) for 24 h, and then OH∙ production in the cells was fluorescently visualized. Detection of hydroxyl radicals in FLS1 cells was performed using a Cell Meter™ mitochondrial hydroxyl radical (OH∙) detection kit under a fluorescence microscope. The nuclei were counter-stained with Hoechst 33342 (blue). Scale bar, 50 μm. Source: <b>Hydrogen peroxide-induced oxidative stress promotes expression of CXCL15/Lungkine mRNA in a MEK/ERK-dependent manner in fibroblast-like synoviocytes derived from mouse temporomandibular joint</b> by Asanuma, Kanna et.al., <em>Journal of Oral Biosciences</em>, March 2023](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcell-meter-mitochondrial-hydroxyl-radical-detection-kit-red-fluorescence%2Ffigure-for-cell-meter-mitochondrial-hydroxyl-radical-detection-kit-red-fluorescence_qhb1D.png&w=3840&q=75)
![Cellular mitochondrial ROS-OH580 activity under 24 h pulsed UV irradiation in MCF-7 and Cos7 cells. (A) The signal intensities of PI and ROS-OH580 in control MCF-7 cells were weakly distributed in the cytoplasm and nucleus. Bar = 5 μm. Cell atrophy and cytoplasmic lysis were observed in irradiated MCF-7 cells. Most cells were PI-positive (cell death), and the ROS-OH580 (•OH) signal was strongly detected in the nuclear enrichment. Bar = 5μm. (B) PI and ROS-OH580 (•OH) signal intensities in Cos7 cells were weakly distributed in the cytoplasm of control and irradiated Cos7 cells. Bar = 5μm. Source: <b>A New Drug-Free Cancer Therapy Using Ultraviolet Pulsed Irradiation. PDT (PhotoDynamic Therapy) to PPT (Pulsed Photon Therapy).</b> by Johbu Itoh, Yoshiko Itoh. <em>Front. Biosci. (Schol Ed)</em>, Sept. 2022.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcell-meter-mitochondrial-hydroxyl-radical-detection-kit-red-fluorescence%2Ffigure-for-cell-meter-mitochondrial-hydroxyl-radical-detection-kit-red-fluorescence_yk6gA.jpeg&w=3840&q=75)
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: Itoh, Johbu and Itoh, Yoshiko
Journal: Frontiers in Bioscience-Scholar (2022): 27
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: 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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Application notes
Abbreviation of Common Chemical Compounds Related to Peptides
Attenuation of lysyl oxidase and collagen gene expression in keratoconus patient corneal epithelium corresponds to disease severity
Copper Transport Protein Antioxidant-1 Promotes Inflammatory Neovascularization via Chaperone and Transcription Factor Function
Dopamine-Mediated Oxidation of Methionine 127 in α-Synuclein