MitoTracker Orange CMTMRos, a fluorescent dye that labels mitochondria in live cells by utilizing the mitochondrial membrane potential (Ex/Em: 551/576 nm) [1].

Labeling of Cells: For live cells, begin with Steps 1–7; for fixed cells, follow Steps 8–13. 1. Prepare a labeling solution with a concentration of 25–500 nM in the selected cell culture medium and warm it to the appropriate culture temperature. 2. Remove the culture medium from cells on coverslips. 3. Submerge the cells in the labeling medium for 15–45 minutes at 37°C. 4. Remove the labeling medium and rinse the cells three times with culture medium. 5. Mount the cells for observation. 6. Examine the cells with a fluorescence microscope. If the fluorescence is inadequate, consider: i. Incubating for an additional 30 minutes in standard culture medium to enhance thiol conjugation. ii. Increasing the initial concentration of the labeling solution. 7. Once sufficient fluorescence is achieved, proceed to image the cells. For fixed cells: 8. Repeat Steps 1–4 with a labeling solution concentration of 100–1000 nM. 9. Wash the cells three times with PBS+. 10. Fix the cells for 10 minutes in 3.7% formaldehyde in PBS (pH 7.4). 11. Aspirate and rinse the cells three times for 5 minutes each with PBS. 12. Mount the coverslips. 13. Image the fixed cells.