The excitation wavelength for MitoSOX Red (Cat. Nos. M36008, M36007, M36009) was changed from 510 nm to 396 nm in the new manual because we have found that excitation at 396 nm selectively excites the superoxide oxidation product without exciting other non-specific products. Although the indicator displays an adequate signal when excited at 510 nm, we highly recommend using the 396 nm excitation wavelength for selective detection of the indicator’s response to mitochondrial superoxide. For more information on the selective detection of superoxide at 396 nm, please refer to the article linked below.
Robinson KM, Janes MS, Pehar M, Monette JS, Ross MF, Hagen TM, Murphy MP, Beckman JS. Selective fluorescent imaging of superoxide in vivo using ethidium-based probes. Proc Natl Acad Sci U S A. 2006 Oct 10;103(41):15038-43. doi: 10.1073/pnas.0601945103. Epub 2006 Oct 2. PMID: 17015830; PMCID: PMC1586181.The optimal emission wavelength to observe the superoxide oxidation product is 580 nm. Please note that the 610 nm wavelength mentioned in the
MitoSOX Green and MitoSOX Red Mitochondrial Superoxide Indicators User Guide is incorrect.
We suggest a working concentration range of 100 nM to 5 µM. It is recommended to optimize the final working concentration for each sample and application. It is important to be cautious when using concentrations >1 µM, as higher non-specific labeling may occur as well as possible mitochondrial toxicity.
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