Babco, Signet, Sternberger Monoclonals, Senetek, Covance

other

JC-10 Mitochondrial Membrane Potential Kit

421902

1 kit

500 USD

421902

JC-10 Mitochondrial Membrane Potential Kit

1 kit

500 USD

Human, Mouse, Rat, All Species

SET

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:;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.;Determine the desired amount of dye loading solution for your assay. 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:;Harvest cells and resuspend in 100 μL of BioLegend Cell Staining Buffer (Cat. No. 420201), PBS, or HBSS at a density of 0.5 - 1x10^6 cells/tube.;Add 50 μL of the prepared dye loading solution to each. This amount works well for most cell types.;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.;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.;Seed cells into chamber slides or well plates with glass coverslip.;Add dye loading solution directly to cells. Volume needed will vary depending on well size. We recommend a dilution of 1 part dye loading solution to 2 parts medium.;Incubate cells for 30-60 min at 37°C protected from light.;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.;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 about 490 nm with a long pass emission filter > 530 nm.Plate Reader Assay:;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.;Add dye loading solution directly to cells. We recommend a dilution of 1 part dye loading solution to 2 parts medium.;Incubate cells for 30-60 min at 37°C protected from light.;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.;Measure fluorescence using a microplate reader for ratiometric analysis. To quantify JC-10 monomer fluorescence, use Ex/Em about 490/525 nm or instrument settings appropriate for fluorescein. To quantify JC-10 aggregate fluorescence, use Ex/Em about 540/590 nm or Texas Red® instrument settings.;