MitoTracker Orange CMTMRos is a live cell fluorescent dye that labels mitochondria in living cells by mitochondrial membrane potential accumulation and is suitable for live cell imaging and apoptosis studies.

Solution preparation:
1. Use DMSO to prepare a certain concentration of stock solution (10 mM), and the working solution is prepared to 10-1000 nM.
Note: Please store the stock solution at -20℃ or -80℃ to avoid repeated freezing and thawing. Please prepare the working solution for immediate use.
Experimental operation steps:
This experiment is divided into live cell and fixed cell labeling. For live cells, please follow steps 1-7, and for fixed cells, please follow steps 8-13.
Cell labeling:
1. Prepare 25-500 nM working solution in the required cell culture medium and warm it to the cell culture temperature.
2. Aspirate the culture medium from the cells growing on the coverslip.
3. Immerse the cells in the labeling medium for 15-45 minutes at 37°C.
4. Aspirate the labeling medium and rinse the cells three times with the medium.
5. Mount the cells.
6. Examine the cells using a fluorescence microscope. If fluorescent staining is too low, try one of the following options:
1) Incubate in normal medium for an additional 30 minutes to allow thiol binding to proceed;
2) Try a higher initial concentration of labeling solution.
7. When fluorescence is sufficient, image the cells.
8. Perform steps 1-4, but use a labeling solution with a working concentration between 100 and 1000 nM.
9. Rinse cells three times with PBS.
10. Fix cells in 3.7% formaldehyde (pH 7.4) in PBS for 10 minutes.
11. Aspirate and rinse cells three times in PBS for 5 minutes each.
12. Mount coverslips.
13. Image the cells.