MitoSOX Red TFA (Mito-hydroethidine TFA) is a fluorescent probe that specifically targets mitochondria in living cells.MitoSOX Red is susceptible to oxidation by superoxide, but is not easily oxidized by other ROS or RNS-generating systems.MitoSOX Red acts as a fluorescent indicator and can be used for live cell imaging.

Instructions
I. Solution preparation
1. Stock solution: MitoSOX Red TFA is usually dissolved in DMSO (dimethyl sulfoxide) to make a stock solution with a common concentration of 1 mM. When storing the stock solution, it should be stored at -20°C away from light and avoid repeated freezing and thawing to maintain stability.
2. Working solution: Dilute the stock solution to the required concentration, generally in the range of 1–5 µM, using an appropriate buffer such as PBS or culture medium. It is recommended to prepare the working solution freshly to ensure the best results.
II. Cell staining steps
1. Cell preparation: For adherent cells, culture the cells on appropriate glass slides or culture plates. For suspended cells, use a suitable culture container.
Make sure the cells are healthy and in the exponential growth phase for the best staining effect.
2. Staining steps:
1) Add the MitoSOX Red working solution directly to the cell culture medium, and the staining time is usually 10–30 minutes. The staining time can be optimized according to the cell type and experimental requirements.
2) During the staining process, avoid light exposure to prevent attenuation of the fluorescence signal.
3) Washing: After staining, remove the dye and wash the cells with PBS or appropriate culture medium to remove unbound dye.
4) Fluorescence microscopy: Observe cells using a fluorescence microscope, with an excitation wavelength of usually 510 nm and an emission wavelength of about 580 nm (red fluorescence), which can be used to observe superoxide in mitochondria.
5) Mitochondrial superoxide detection MitoSOX Red can be used to assess mitochondrial oxidative stress and superoxide production; the fluorescence intensity is related to the level of superoxide in mitochondria, so it can be used to study mitochondrial dysfunction, oxidative stress and related pathological processes.
Precautions
1. Photosensitivity: MitoSOX Red is sensitive to light and should be avoided from prolonged exposure to strong light and stored in a light-proof environment to prevent attenuation of the fluorescence signal.
2. Toxicity: Use appropriate concentrations to avoid cytotoxicity. MitoSOX Red is generally safe at recommended concentrations, but excessive concentrations may cause cell damage, especially in long-term experiments.
3. Storage: MitoSOX Red stock solution should be stored at -20°C in the dark. Avoid repeated freezing and thawing as this may affect the stability and performance of the probe.
4. Mitochondrial membrane potential: Changes in mitochondrial membrane potential can affect the uptake of MitoSOX Red. Therefore, normal mitochondrial membrane potential should be ensured before the experiment to ensure effective staining.