JC-1 Mitochondrial Membrane Potential Dye

1. Prepare cells with test compounds
2.  Add JC-1 working solution (100 µL/well for 96-well plates or 25 µL/well for 384-well plates)
3. Incubate at room temperature or 37°C for 1 hr
4. Remove the JC-1 working solution
5. Read fluorescence intensity at Ex/Em = 490/525 nm and 490/590 nm

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

Prepare a 2 to 10 mM stock solution of JC-1 in high-quality, anhydrous DMSO. The stock solution should be used promptly; any remaining solution should be aliquoted and frozen at < -20°C.

Note        Avoid repeated freeze-thaw cycles, and protect from light. 

Prepare a 1X JC-1 working solution: On the day of the experiment, either dissolve JC-1 solid in DMSO or thaw an aliquot of the JC-1 stock solution to room temperature. Prepare a 10 to 30 µM 1X JC-1 working solution in Hanks and 20 mM Hepes buffer (HHBS) or buffer of your choice, pH 7 with 0.02% Pluronic® F-127. Mix them well by votexing. 

Note        JC-1is not water soluble, so it intends to aggregate in solution. It is recommended to filter the JC-1 working solution before loading it into the cells.

1. Treat cells with test compounds for a desired period of time (For example, Jurkat cells can be treated with camptothecin for 4-6 hours) to induce apoptosis. For blank wells (medium without the cells), add the corresponding amount of compound buffer.
2. Add 100 µL/well/96-well plate or 25 µL/well/384-well plate of JC-1 working solution (from Step 1) into the cell plate.
3. Incubate the JC-1 loading plate in a 37°C, 5% CO2 incubator for 15-60 min.
   
   Note        The appropriate incubation time depends on the individual cell type and cell concentration used.
   Optimize the incubation time for each experiment.
4. Remove the JC-1 working solution from the plate, wash the cells with HHBS or buffer of your choice. Add 100 µL/well/96-well plate or 25 µL/well/384-well plate of HHBS back to the cell plate.
5. Monitor the fluorescence change at Ex/Em = 490/525 nm and 490/590 nm for ratio analysis.

1. Treat cells with test compounds for a desired period of time (For example, Jurkat cells can be treated with camptothecin for 4-6 hours) to induce apoptosis. 
2. Centrifuge the cells to get 1-5 × 105 cells per tube. 
3. Resuspend cells in 500 μL of JC-1 working solution (from Step 1). 
4. Incubate at room temperature or 37 °C for 10 to 30 min, protected from light.
5. Wash the cells with HHBS or buffer of your choice. Resuspend cells in 500 μL of HHBS to get 1-5 × 10⁵ cells per tube.
6. Monitor the fluorescence change at Ex/Em = 490/525 nm and 490/590 nm with a fluorescence microscope (using FITC and TRITC filters) or a flow cytometer (using FL1 and FL2 channels).