Cell Meter™ Mitochondrion Membrane Potential Assay Kit *Red Fluorescence Optimized for Flow Cytometry*

Protocol summary

1. Prepare cells with test compounds at the density of 5 × 10⁵ to 1 × 10⁶ cells/mL
2. Add 1 µL of 500X MitoTell™ Red into 0.5 mL of cell solution
3. Incubate the cells in a 37 °C, 5% CO₂ incubator for 15 - 30 minutes
4. Pellet the cells, and resuspend the cells in 0.5 mL of assay buffer
5. Analyze cells using flow cytometer with APC or Cy5 channel

Important notes
Thaw all the kit components at room temperature before starting the experiment.

1. For each sample, prepare cells in 0.5 mL of warm medium or buffer of your choice at the density of 5×10⁵ to 1×10⁶ cells/mL. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for apoptosis induction.
   
   
2. Treat cells with test compounds for a desired period of time to induce apoptosis, and set up parallel control experiments. Note: We treated Jurkat cells with 20µM CCCP for 15 min at 37ºC to change the mitochondrial membrane potential. See Figure 1 for details. CCCP or FCCP can be added simultaneously with MitoTell™ Red. To get the best result, titration of the CCCP or FCCP may be required for each individual cell line.
   
   
3. Add 1 µL of 500X MitoTell™ Red (Component A) into the treated cells.
   
   
4. Incubate the cells in a 37 °C, 5% CO₂ incubator for 15 to 30 minutes. Note: For adherent cells, gently lift the cells with 0.5 mM EDTA to keep the cells intact and wash the cells once with serum-containing media prior to the incubation with MitoTell™ Red.
   
   
5. Centrifuge the cells at 800 rpm for 4 minutes, and then re-suspend cells in 0.5 mL of Assay Buffer (Component B) or buffer of your choice.
   
   
6.  Monitor the fluorescence intensity using a flow cytometer wih APC or Cy5 channel. Gate on the cells of interest, excluding debris.