Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit *Green Fluorescence*
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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) | 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 | SDSProtocol |
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
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: Slocinska M, Rosinski G, Jarmuszkiewicz W.
Journal: Protein Pept Lett (2016): 63
Authors: Wu Y, Shamoto-Nagai M, Maruyama W, Osawa T, Naoi M.
Journal: J Neural Transm (Vienna) (2016): 491
Authors: Ekiert R, Borek A, Kuleta P, Czernek J, Osyczka A.
Journal: Biochim Biophys Acta (2016): 1102
Authors: Achour I, Arel-Dubeau AM, Renaud J, Legr and M, Attard E, Germain M, Martinoli MG.
Journal: Int J Mol Sci (2016): 1293
Authors: Govatati S, Malempati S, Saradamma B, Divyamaanasa D, Naidu BP, Bramhachari PV, Narayana N, Shivaji S, Bhanoori M, Tamanam RR, Rao PS, Nallanchakravarthula V.
Journal: Tumour Biol. (2016)
Authors: Goncalves RL, Bunik VI, Br and MD., undefined
Journal: Free Radic Biol Med (2016): 247
Authors: Chouchani ET, Pell VR, James AM, Work LM, Saeb-Parsy K, Frezza C, Krieg T, Murphy MP.
Journal: Cell Metab (2016): 254
Authors: Ochi R, Dhagia V, Lakhkar A, Patel D, Wolin MS, Gupte SA.
Journal: Am J Physiol Heart Circ Physiol (2016): H1118
Authors: Ciarcia R, Damiano S, Squillacioti C, Mirabella N, Pagnini U, Florio A, Severino L, Capasso G, Borrelli A, Mancini A, Boffo S, Romano G, Giordano A, Florio S.
Journal: J Cell Biochem (2016): 1352
Authors: Ni R, Zheng D, Xiong S, Hill DJ, Sun T, Gardiner RB, Fan GC, Lu Y, Abel ED, Greer PA, Peng T.
Journal: Diabetes (2016): 255
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