Cell Meter™ JC-10 Mitochondrion Membrane Potential Assay Kit *Optimized for Flow Cytometry Assays*

Protocol summary

1. Prepare cells with test compounds at the density of 5 × 10⁵ to 1 x 10⁶ cells/mL
2. Resuspend the cells in 500 µL of JC-10 working solution (2-5 × 10⁵ cells/tube) 
3. Incubate at 37°C or room temperature for 15-60 minutes
4. Analyze with flow cytometer using FL1 channel (green fluorescence monomeric signal) and FL2 channel (orange fluorescence aggregated signal)

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

Add 25 µL of 200X JC-10 (Component A) into 5 mL of Assay Buffer A (Component B) and mix well to make JC-10 working solution. Protect from light.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

1. Treat cells with test compounds for a desired period of time to induce apoptosis. Set up parallel control experiments.
   
   For Negative Control: Treat cells with vehicle only.
   
   For Positive Control: Treat cells with FCCP or CCCP at 2-10  µM in a 37 oC, 5% CO2 incubator for 15 to 30 minutes. Note: CCCP or FCCP can be added simultaneously with JC-10 working solution. Titration of the CCCP or FCCP may be required for optimal results with an individual cell lines. 
   
   
2. Centrifuge the cells to get 2 - 5 × 10⁵ cells per tube. Note: For adherent cells, gently lift the cells by 0.5 mM EDTA to remain the cells intake, and wash the cells once with serum-containing media prior to incubation with JC-10 working solution.
   
   
3. Resuspend cells in 500 µL of JC-10 working solution.
   
   
4. Incubate the cells at room temperature or in a 37 oC, 5% CO2 incubator for 15 - 60 minutes, protected from light. Note: The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment.
   
   
5. Monitor the fluorescence intensity using flow cytometer with FL1 channel for the green fluorescence monomeric signal (in apoptotic cells), and FL2 channel for the orange fluorescence aggregated signal (in healthy cells). Gate on the cells, excluding debris. It is recommended that compensation corrections be performed using the FCCP or CCCP-treated cells.

• Typical Flow Cytometer settings for the analysis of JC-10 on a BD FACS Calibur System flow cytometer are as follows:
  Suggested initial conditions may require modifications because of differences in cell types and culture conditions, and also the individual instrumentation.
  FL1 PMT voltage 366 
  FL2 PMT voltage 430
  Compensation: FL1 – 47.2% FL2; FL2 – 47.0% FL1