MitoLite™ Green FM

Protocol Summary

1. Prepare 1 mM MitoLite™ Green FM stock solution
2. Prepare 20-200 nM MitoLite™ Green FM staining solution
3. Remove the growth media from the cells
4. Add MitoLite™ Green FM staining solution to cells
5. Incubate at 37°C for 30 minutes
6. Wash cells and replace with 1x Hanks and 20mM Hepes Buffer (HH buffer)
7. Observe cells using a fluorescence microscope with FITC filter set

Important  Bring the dye at room temperature before starting the experiment.

MitoLite™ Green FM stock solution:
Dissolve one vial MitoLite™ Green FM (50 ug) in 74 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™ Green FM staining solution:
Dilute 1 mM MitoLite™ Green FM stock solution to the final working concentration in HH buffer. The working concentration can be in the range of 20–200 nM.

Staining adherent cells:

1. Grow cells to reach the desired confluency.
   
   
2. Remove the growth media from the cells.
   
   
3. Add MitoLite™ Green FM 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 FITC 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™ Green FM 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 flow cytometry (530/30 nm filter- FITC channel) or fluorescence microscopy (FITC filter set).