JC-10 *Superior alternative to JC-1*

1. Prepare cells with test compounds
2. Add JC-10 working solution (100 µL/well for 96-well plates or 25 µL/well for 384-well plate)
3. Incubate at room temperature or 37°C for 1 hr
4. Read fluorescence intensity at Ex/Em = 490/525 nm and 540/590 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

Each vial of DMSO stock solution (100 µL, 2 mg/mL, 3 mM) should be used only once. Any unused vials should be stored at < -20°C.

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

Prepare a 1X JC-10 working solution: On the day of the experiment, thaw an aliquot of the JC-10 stock solution to room temperature. Prepare a 10 to 30 µM 1X working solution in Hanks and 20 mM Hepes buffer (HHBS) or buffer of your choice, pH 7-8 with 0.02% Pluronic® F-127. Mix them well by votexing.

Note        For some cell lines, working solution at pH 8 might prevent JC-10 leakage. 

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-10 working solution (from Step 1) into the cell plate.
3. Incubate the JC-10 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. Monitor the fluorescence change at Ex/Em = 490/525 nm (FITC channel) and 540/590 nm (TRITC channel) for ratio analysis.
   
   Optional: Remove the JC-10 working solution from the plate; add 100 µL/well/96-well plate or 25 µL/well/384-well plate of HHBS back to the cell plate before 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-10 working solution (from Step 2)
4. Incubate at room temperature or in a 37 °C, 5%CO2 incubator for 10 to 30 min, 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 change at Ex/Em = 490/525 nm and 540/590 nm with a fluorescence microscope (using FITC and TRITC filters) or a flow cytometer (using FL1 and FL2 channels).
   
   Optional: Remove the JC-10 working solution from the plate; add 100 µL/well/96-well plate or 25 µL/well/384-well plate of HHBS back to the cell plate before analysis on fluorescence microscope.