MitoLite™ Red CMXRos

Protocol Summary

1. Prepare 1 mM MitoLite™ Red CMXRos stock solution
2. Prepare 100-500 nM MitoLite™ Red CMXRos staining solution
3. Remove the growth media from the cells
4. Add MitoLite™ Red CMXRos staining solution to cells
5. Incubate at 37°C for 30 minutes
6. Wash cells and repalce with 1x Hanks and 20mM Hepes Buffer (HH buffer)
7. Observe cells using a fluorescence microscope with TRITC filter set

MitoLite™ Red CMXRos stock solution:
Dissolve one vial MitoLite™ Red CMXRos (50 ug) in 94 uL high-quality, anhydrous dimethylsulfoxide (DMSO) to make 1 mM stock solution. Note: Keep the stock solution frozen at ≤–15°C and protected from light.

MitoLite™ Red CMXRos staining solution:
Dilute 1 mM MitoLite™ Red CMXRos stock solution to the final working concentration in HH buffer. Note: The working concentration can be in the range of 100–500 nM.

Staining adherent cells:

1. Grow cells to reach the desired confluency.
   
   
2. Remove the growth media from the cells.
   
   
3. Add MitoLite™ Red CMXRos staining solution to each well.
   
   
4. Incubate at 37°C for 30 minutes.
   
   
5. Wash cells and replace with 1x Hanks and 20mM Hepes Buffer (HH buffer).
   
   
6. Observe cells using a fluorescence microscope with TRITC filter set. Note: The staining protocols is good for HeLa cell line and it may need to be optimized with the particular cell types.

Staining suspension cells:

1. Centrifuge cells to a pellet and aspirate the supernatant.
   
   
2. Resuspend the cells gently in MitoLite™ Red CMXRos staining solution.
   
   
3. Incubate at 37°C for 30 minutes.
   
   
4. Centrifuge the cells, remove supernatant and resuspend cells in fresh HH buffer.
   
   
5. Cells may be analyzed by fluorescence microscopy (TRITC filter set).