1. After receiving the dye, store it at -20 ℃. When using it, dissolve 100 μg MitoSOX Red with 26 μL DMSO. Dissolve it at 4 ℃ each time you use it, and prepare it into a working solution at a ratio of 1:500.
2. The stained cells are single-cell suspensions extracted from tissues (C57 mouse hippocampus), which are HL-1 cardiomyocytes, made from fresh tissues, with a cell count of 5-6*10^5/mL. When staining, take 200 μL of cell suspension, centrifuge to keep the precipitate, add 200 μL MitoSOX Red working solution for staining, and incubate at room temperature for 20 min.
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Preparation method of working solution:
According to the instructions on the official website, it is recommended to weigh 100 μg of powder and add 26 μL DMSO, dissolve it and then divide it, and directly dilute it with the corresponding working solution for staining. The remaining divided dyes are directly stored in a -80℃ refrigerator away from light. The concentration of the obtained working solution is 5 μM. When treating the HL-1 cardiomyocyte cell line, first treat it with doxorubicin (1 μM) for 24h.
Staining method:
MitoSOX Red needs to enter the cell through the cell membrane and depends on the mitochondrial membrane potential to be enriched in the mitochondria. Incubation at 37℃ is more in line with the physiological state of the cell and can promote active uptake of the dye; while incubation at room temperature (20-25℃) may reduce the metabolic activity of the cell, resulting in a decrease in mitochondrial uptake efficiency and a weakening of the fluorescence signal. Recommendation: Adjust the incubation conditions to 37℃, 5% CO₂ incubator away from light for 15-20min to improve the targeting of the dye.
Moreover, the fluorescent product of MitoSOX Red is extremely sensitive to light. The entire process from dye dissolution to staining, centrifugation, and machine detection must be strictly protected from light (such as wrapping the container with tin foil), otherwise the fluorescence will be easily quenched and the signal will be weakened.
The answer to this question comes from MedChemExpress (MCE) Technical Support.
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