Hello Chloe Sun,

MitoSOX Red is a positively charged molecule that accumulates in mitochondria due to the negatively charged mitochondrial matrix. Once inside, it reacts with superoxide and is oxidized to a red-fluorescent product, ethidium. This ethidium, derived from MitoSOX Red oxidation, can diffuse out of the mitochondria and bind to DNA in the nucleus, causing nuclear staining. 

When MitoSOX Red is used at higher concentrations, it can start to accumulate in the nucleus. Also, damaged mitochondria can release superoxide and the dye into the cytoplasm, potentially leading to increased staining in other cellular compartments, including the nucleus. 

Therefore, it is crucial to optimize the concentration of MitoSOX Red in order to minimize non-specific staining. While MitoSOX Red is a common dye for measuring superoxide in mitochondria, there are alternatives. But when choosing an alternative, you should consider the specific ROS you are interested in, the desired level of specificity for mitochondria, and potential limitations of each probe.

Best,

Following cellular uptake, MitoSOX™ Red (HY-D1055, MedChemExpress) is selectively taken up by mitochondria and localized within the organelle. The probe itself is a non-fluorescent precursor. Once inside the mitochondria, MitoSOX™ Red undergoes specific oxidation by superoxide anion (O₂⁻), generating a fluorescent product. This oxidized product subsequently binds to nucleic acids. It is important to note that this binding occurs not only within mitochondria but also in the nucleus. Consequently, the intensity of the fluorescence signal serves as an indicator for relative cellular superoxide anion levels, primarily reflecting mitochondrial O₂⁻ production.

The answer to this question comes from MedChemExpress (MCE) Technical Support.

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