MitoTracker Orange CMTMRos

• CAT Number: I043051
• CAS Number: 199116-50-2
• Molecular Formula: C24H24Cl2N2O
• Molecular Weight: 427.37
• Purity: ≥95%
• Synonyms: [9-[4-(chloromethyl)phenyl]-6-(dimethylamino)xanthen-3-ylidene]-dimethylazanium;chloride
• InChI: InChI=1S/C24H24ClN2O.ClH/c1-26(2)18-9-11-20-22(13-18)28-23-14-19(27(3)4)10-12-21(23)24(20)17-7-5-16(15-25)6-8-17;/h5-14H,15H2,1-4H3;1H/q+1;/p-1
• InChIKey: FZTMEYOUQQFBJR-UHFFFAOYSA-M
• SMILES: CN(C)C1=CC2=C(C=C1)C(=C3C=CC(=[N+](C)C)C=C3O2)C4=CC=C(C=C4)CCl.[Cl-]

MitoTracker Orange CMTMRos is a fluorescent dye that labels mitochondria within live cells utilizing the mitochondrial membrane potential (Ex/Em: 551/576 nm)[1].
Guidelines[1] (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs).
Labeling of Cells:
For live cells, follow Steps 1–7. For fixed cells, follow Steps 8–13.
1. Prepare a labeling solution at 25–500 nM in the desired cell culture medium, and warm it to the cell culture temperature.
2. Aspirate the culture medium from cells grown on coverslips.
3. Immerse the cells in the labeling medium for 15–45 min at 37?C.
4. Aspirate the labeling medium, and rinse the cells three times with culture medium.
5. Mount the cells.
6. Check the cells using a fluorescence microscope. If the fluorescence staining is too low, try one of the following options:
i. Incubate for an additional 30 min in the normal culture medium to allow the thiol conjugation to proceed.
ii. Try a higher initial concentration of the labeling solution.
7. When the fluorescence is sufficient, image the cells.
8. Carry out Steps 1–4, but use a labeling solution at a working concentration between 100 and 1000 nM.
9. Rinse the cells three times with PBS+.
10. Fix the cells for 10 min in 3.7% formaldehyde in PBS (pH 7.4).
11. Aspirate and rinse the cells three times for 5 min each in PBS.
12. Mount the coverslips.
13. Image the cells.

Reference

[1]. Chazotte B. Labeling mitochondria with MitoTracker dyes. Cold Spring Harb Protoc. 2011 Aug 1;2011(8):990-2.
 [Content Brief]