JC-10 Mitochondrial Membrane Potential Kit

• 100X JC-10 in DMSO (~3.5 mM) – 250 μL
• Dye Loading Buffer – 25 mL
• Masking Solution – 25 mL

Please refer to detailed protocol in application notes

General Considerations:

• Optimal dye concentration and loading time will vary depending on cell type and application. Recommended dye concentrations range between 1-15 μM JC-10.
• JC-10 is compatible with kinetic imaging and analysis for up to 8 hrs after JC-10 addition under the appropriate experimental conditions.
• Carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP) and carbonylcyanide-3-chlorophenylhydrazone (CCCP) are potent mitochondrial uncouplers which can be used for positive controls. An effective FCCP and CCCP concentration is typically 2-5 μM for 30 minutes at 37°C prior to dye addition for most cell types.

Reagent preparation:

1. Allow all reagents to thaw and reach room temperature protected from light.
   Note: Repeated freeze/thaw cycles of the JC-10 dye may cause loss of performance. We recommend storing the dye in one time use aliquots at -20°C.
2. Determine the desired amount of dye loading solution for your assay. 
3. Prepare the dye loading solution by diluting the 100X JC-10 stock solution 100-fold with the dye loading buffer. For example, add 10 μL JC-10 stock solution to 1 mL of dye loading buffer. Vortex until solution transitions from pink to near-colorless.

Flow Cytometry Assay:

1. Harvest cells and resuspend in 100 μL of BioLegend Cell Staining Buffer (Cat. No. 420201), PBS, or HBSS at a density of 0.5 - 1x10⁶ cells/tube.
2. Add 50 μL of the prepared dye loading solution to each. This amount works well for most cell types.
3. Incubate at room temperature or 37°C for 15-30 minutes.
   Optional: Add up to 400 μL of additional buffer to adjust cell concentration before running on cytometer.
4. Analyze cells using a flow cytometer using the PE and FITC channels.

Note: For adjusting compensation, use FCCP/CCCP treated positive controls, which will have strong fluorescence in FITC channel with little or no signal in PE channel.

Note: Samples with healthy mitochondria will be positive in both channels but will have a stronger signal in the PE channel than FCCP/CCCP treated samples.

Microscopy Assay:

It is possible to load JC-10 dye prior to the addition of test compounds for real-time visualization of mitochondrial membrane depolarization. Time-lapse imaging of cells can continue for up to 8 hours after dye addition.

1. Seed cells into chamber slides or well plates with glass coverslip.
2. Add dye loading solution directly to cells. Volume needed will vary depending on well size. We recommend a dilution of 1:2 loading dye solution: medium.
3. Incubate cells for 30-60 min at 37°C protected from light.
4. Add masking solution directly to cells. Volume needed will vary depending on well size. We recommend a dilution of 1 part masking solution to 3 parts total well volume. Do not remove dye loading solution from wells.
5. Image cells using an inverted fluorescence microscope. JC-10 monomers can be visualized with standard FITC or GFP filters, and JC-10 aggregates can be viewed using TRITC or Texas Red® filters.

Note: Optimal imaging setup to simultaneously capture monomers and aggregates would be to use an excitation filter of ~490 nm with a long pass emission filter > 530 nm.

Plate Reader Assay:

1. Seed cells into a black 96-well plate and treat with test compounds of your choosing prior to the addition of dye loading solution. Final volume should be 100 μL/well.
2. Add dye loading solution directly to cells. We recommend a dilution of 1 part dye loading solution to 2 parts medium.
3. Incubate cells for 30-60 min at 37°C protected from light.
4. Add masking solution directly to cells. Volume needed will vary depending on well size. We recommend a dilution of 1 part masking solution to 3 parts volume in well. Do not remove dye loading solution from wells.
5. Measure fluorescence using a microplate reader for ratiometric analysis. To quantify JC-10 monomer fluorescence, use Ex/Em ~490/525 nm or instrument settings appropriate for fluorescein. To quantify JC-10 aggregate fluorescence, use Ex/Em ~540/590 nm or Texas Red® instrument settings.

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Mitochondrial matrix, cytoplasm