Cell Meter™ Mitochondrion Membrane Potential Assay Kit *Orange 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 2 µL of 500X MitoTell™ Orange into 1 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 1 mL of growth medium
5. Analyze cells using flow cytometer with FL2 channel

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

1. For each sample, prepare cells in 1 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.
   
   For Negative Control: Treat cells with vehicle only.
   
   For Positive Control: Treat cells with FCCP or CCCP at 5-50 µM in a 37 oC, 5% CO₂ incubator for 15 to 30 minutes. Note: CCCP or FCCP can be added simultaneously with MitoTell™ Orange. To get the best result, titration of the CCCP or FCCP may be required for each individual cell line.
   
   
3. Add 2 µL of 500X MitoTell™ Orange (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™ Orange dye-loading solution.
   
   
5. Centrifuge the cells at 1000 rpm for 4 minutes, and then re-suspend cells in 1 mL of Assay Buffer (Component B) or buffer of your choice.
   
   
6.  Monitor the fluorescence intensity using a flow cytometer wih FL2 channel (Ex/Em = 540/590 nm). Gate on the cells of interest, excluding debris.