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

Protocol summary

1. Prepare cells
2. Add test compounds
3. Add JC-10 dye-working solution (50 µL/well/96-well plate or 12.5 µL/well/384-well plate) 
4. Incubate at 37°C, 5% CO₂ incubator for 30 to 60 minutes
5. Add Assay Buffer B (50 µL/well/96-well plate or 12.5 µL/well/384-well plate)
6. Monitor fluorescence intensities (bottom read mode) at Ex/Em = 490/525 nm (Cutoff = 515 nm) and 540/590 nm (Cutoff = 570 nm)

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

Add 50 µL of 100X JC-10 (Component A) into 5 mL of Assay Buffer A (Component B) and mix well to make JC-10 dye-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 by adding 10 µL of 10X test compounds (96-well plate) or 5 µL of 5X test compounds (384-plate) into the desired buffer (such as PBS or HHBS). Note: It is not necessary to wash cells before adding compound. However, if tested compounds are serum sensitive, growth medium and serum factors can be aspirated away before adding compounds. Add the same volume of HHBS into the wells (such as 90 µL for a 96-well plate or 20 µL for a 384-well plate) after aspiration. Alternatively, cells can be grown in serum-free media.
   
   
2. Incubate the cell plate at room temperature or in a 37°C, 5% CO₂ incubator for at least 15 minutes or a desired period of time (for Jurkat cells, 4 - 6 hours with camptothecin or 3 - 5 hours with staurosporine treatment) to induce apoptosis.
   
   
3. Add 50 µL/well (96-well plate) or 12.5 µL/well (384-well plate) of JC-10 dye-working solution into the cell plate.
   
   
4. Incubate the plate in a 37°C, 5% CO₂ incubator for 30 - 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. Add 50 µL/well (96-well plate) or 12.5 µL/well (384-well plate) of Assay Buffer B (Component C) into JC-10 dye-working solution plate before reading the fluorescence intensity. Note: DO NOT wash the cells after loading. For non-adherent cells, it is recommended to centrifuge cell plates at 800 rpm for 2 minutes with brake off after adding Assay Buffer B (Component C).
   
   
6. Monitor the fluorescence intensities with a fluorescence microplate reader (bottom read mode) at Ex/Em = 490/525 nm (Cutoff = 515 nm) and 540/590 nm (Cutoff = 570 nm) for ratio analysis.