Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit *Green Fluorescence*
Price | |
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) | 513 |
Emission (nm) | 537 |
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 | 12352200 |
Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit*Optimized for Flow Cytometry* |
Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit*Optimized for Microplate Reader* |
Overview | ![]() ![]() |
Excitation (nm) 513 | Emission (nm) 537 |
Platform
Flow cytometer
Excitation | 488 nm laser |
Emission | 530/30 nm filter |
Instrument specification(s) | FITC channel |
Fluorescence microscope
Excitation | FITC filter set |
Emission | FITC filter set |
Recommended plate | Black wall/clear bottom |
Instrument specification(s) | FITC filter set |
Components
Example protocol
AT A GLANCE
Protocol summary (Fluorescence Microscope)
- Prepare cells in growth medium
- Treat the cells with test compounds to induce superoxide
- Add MitoROS™ 520 working solution
- Incubate the cells at 37°C for 1 hour
- Monitor the fluorescence using FITC fliter set
Protocol summary (Flow Cytometry)
- Prepare cells in growth medium
- Treat the cells with test compounds to induce superoxide
- Add MitoROS™ 520 stock solution and incubate the cells at 37°C for 1 hour
- Monitor the fluorescence intensity with a flow cytometer using 530/30 nm filter (FITC channel)
Important notes
Thaw all the components at room temperature before use.
PREPARATION OF STOCK SOLUTION
1. MitoROS™ 520 stock solution (500X):
Add 50 µL of DMSO (Component C) into the vial of MitoROS™ 520 (Component A) and mix well. Note: 25 µL of reconstituted MitoROS™ 520 stock solution is enough for 1 plate. Note: Unused portion can be aliquoted and stored at < -20 °C for more than one month if the tubes are sealed tightly and kept from light. Avoid repeated freeze-thaw cycles.
PREPARATION OF WORKING SOLUTION
Only for Fluorescence Microscope
Add 5 μL of 500X DMSO reconstituted MitoROS™ 520 stock solution into 2 mL of Assay Buffer (Component B) and mix well. Note: This working solution is not stable and needs to be prepared freshly before use.
For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html
SAMPLE EXPERIMENTAL PROTOCOL
For Fluorescence Microscopes/96-Well Microplates:
- Treat cells with 10 µL of 10X test compounds (96-well plate) or 5 µL of 5X test compounds (384-well plate) in medium or your desired buffer (such as PBS or HHBS). For control wells (untreated cells), add the corresponding amount of compound buffer.
- To induce superoxide, incubate the cell at 37°C for a desired period of time, protected from light. Note: We treated RAW 264.7 macrophage cells with 5 µM Antimycin A (AMA) at 37°C for 2 hours to induce superoxide. See Figure 1 for details.
- Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of MitoROS™ 520 working solution into the cell plate.
- Incubate the cells at 37°C for 1 hour, and take images using fluorescence microscope with a FITC filter set.
For Flow Cytometers:
- Treat cells as desired.
- To induce superoxide, incubate the cells at 37°C for a desired period of time, protected from light. Note: We treated Jurkat cells with 50 µM Antimycin A (AMA) at 37°C for 2 hours to induce superoxide.
- Add 1 µL/0.5 mL cells of MitoROS™ 520 stock solution (500X) into the cells.
- Incubate the cells in a 5% CO2, 37°C incubator for 1 hour, and monitor the fluorescence intensity using a flow cytometry with 530/30 nm filter (FITC channel).
Images
![Fluorescence images of superoxide measurement in macrophage cells using cat#16060. RAW 264.7 cells at 100,000 cells/well/100 µL were seeded overnight in a 96-well black wall/clear bottom plate. AMA Treatment: Cells were treated with 5 µM Antimycin A (AMA) at 37 °C for 2 hours, then incubated with MitoROS™ 520 for 1 hour. Untreated Control: RAW 264.7 cells were incubated with MitoROS™ 520 at 37 °C for 1 hour without AMA treatment. The fluorescence signal was measured using fluorescence microscope with a FITC filter](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcell-meter-fluorimetric-mitochondrial-superoxide-activity-assay-kit-green-fluorescence%2Ffigure-for-cell-meter-fluorimetric-mitochondrial-superoxide-activity-assay-kit-green-fluorescence_ac1Gi.jpg&w=3840&q=75)
![Fluorescence images of endogenous superoxide measurement in macrophage cells using Cat#16060. RAW 264.7 cells at 100,000 cells/well/100 µL were seeded overnight in a 96-well black wall/clear bottom plate. LPS Treatment: Cells were incubated with MitoROS™ 520 for 1 hour, then treated with 200 μg/mL of lipopolysaccharide (LPS) at 37 ºC for 16 hours. Untreated Control: RAW 264.7 cells were incubated with MitoROS™ 520 at 37 ºC for 1 hour without LPS treatment. The fluorescence signal was measured using fluorescence microscope with a FITC filter.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcell-meter-fluorimetric-mitochondrial-superoxide-activity-assay-kit-green-fluorescence%2Ffigure-for-cell-meter-fluorimetric-mitochondrial-superoxide-activity-assay-kit-green-fluorescence_DGUXz.jpg&w=3840&q=75)
Citations
Authors: Akinci, Ersin and Cha, Minsun and Lin, Lin and Yeo, Grace and Hamilton, Marisa C and Donahue, Callie J and Bermudez-Cabrera, Heysol C and Zanetti, Larissa C and Chen, Maggie and Barkal, Sammy A and others,
Journal: BioRxiv (2020)
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
Authors: Abe, Naoki and Choudhury, Mohammed E and Watanabe, Minori and Kawasaki, Shun and Nishihara, Tasuku and Yano, Hajime and Matsumoto, Shirabe and Kunieda, Takehiro and Kumon, Yoshiaki and Yorozuya, Toshihiro and others, undefined
Journal: Glia (2018)
References
Authors: Demaurex N, Schwarzl and er M., undefined
Journal: Antioxid Redox Signal. (2016)
Authors: Abdukeyum GG, Owen AJ, Larkin TA, McLennan PL.
Journal: J Clin Med (2016): 32
Authors: Alachkar H, Fulton N, Sanford B, Malnassy G, Mutonga M, Larson RA, Bloomfield CD, Marcucci G, Nakamura Y, Stock W.
Journal: Pharmacogenomics J. (2016)
Authors: Han YH, Buffolo M, Pires KM, Pei S, Scherer PE, Boudina S.
Journal: Diabetes (2016): 2639
Authors: Br, undefined and MD., undefined
Journal: Free Radic Biol Med. (2016)
Authors: Huang Y, Yu F, Wang J, Chen L.
Journal: Anal Chem (2016): 4122
Authors: Patel D, Alhawaj R, Kelly MR, Accarino JJ, Lakhkar A, Gupte SA, Sun D, Wolin MS.
Journal: Am J Physiol Heart Circ Physiol (2016): H1439
Authors: Guidarelli A, Fiorani M, Carloni S, Cerioni L, Balduini W, Cantoni O.
Journal: Toxicol Appl Pharmacol (2016): 35
Authors: Lakhkar A, Dhagia V, Joshi SR, Gotlinger K, Patel D, Sun D, Wolin MS, Schwartzman ML, Gupte SA.
Journal: Am J Physiol Heart Circ Physiol (2016): H1107
Authors: Duan Q, Wang T, Zhang N, Perera V, Liang X, Abeysekera IR, Yao X.
Journal: Endocrinol Metab (Seoul) (2016): 174
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