MitoSOX Red is a live cell fluorescent probe that specifically targets mitochondria and is cell membrane permeable. MitoSOX Red enters mitochondria and is oxidized by superoxide but not by other ROS or RNS generating systems. The oxidized MitoSOX Red then binds to nucleic acids in mitochondria/nucleus, producing strong red fluorescence. MitoSOX Red can be used as a fluorescent indicator to specifically detect superoxide. In addition, superoxide dismutase (SOD) can prevent the oxidation of MitoSOX Red.
Excitation/emission wavelength: 510/580 nm.

General Protocol

1. Preparation of MitoSOX Red working solution
1.1 Preparation of the stock solution
Dissolve 50 μg MitoSOX Red in 13 μL DMSO to obtain 5 mM of stock solution.
Note: It is recommended to store the stock solution at -20°C and -80°C away from light and avoid repetitive freeze-thaw cycles.
1.2 Preparation of MitoSOX Red working solution
Dilute the stock solution in serum-free cell culture medium or PBS to obtain 1-10 μM of working solution.
Note: Please adjust the concentration of MitoSOX Red working solution according to the actual situation.
2. Cell staining
2.1 Suspension cells（6-well plate）
a. Centrifuge at 1000 g at 4°C for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time.The cell density is 1×10⁶/mL.
b. Add 1 mL of working solution, and then incubate at room temperature for 5-30 minutes.
c. Centrifuge at 400 g at 4°C for 3-4 minutes and then discard the supernatant.
d. Wash twice with PBS, 5 minutes each time.
e. Resuspend cells with serum-free cell culture medium or PBS. Observation by fluorescence microscopy or flow cytometry.
2.2 Adherent cells
a. Culture adherent cells on sterile coverslips.
b. Remove the coverslip from the medium and aspirate excess medium.
c. Add 100 μL of working solution, gently shake it to completely cover the cells,and then incubate at room temperature for 5-30 minutes.
d. Wash twice with medium, 5 minutes each time. Observation by fluorescence microscopy or flow cytometry.

Storage
-20°C, 1 year. Protect from light

Precautions
1. Please adjust the concentration of MitoSOX Red working solution according to the actual situation.
2. This product is for R&D use only, not for drug, household, or other uses.
3. For your safety and health, please wear a lab coat and disposable gloves to operate.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light, stored under nitrogen). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. OT, et al. Quantitative fluorescence of 5-FU-treated fetal rat limbs using confocal laser scanning microscopy and Lysotracker Red. Cytometry A. 2003;53(1):9-21.  [Content Brief]

  [2]. Fengjiao Jin, et al. The PI3K/Akt/GSK-3β/ROS/eIF2B pathway promotes breast cancer growth and metastasis via suppression of NK cell cytotoxicity and tumor cell susceptibility. Cancer Biol Med. 2019 Feb;16(1):38-54.  [Content Brief]