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| Product Name | Mitochondrial Membrane Potential JC-10 Assay Kit, Flow Cytometry & Fluorescence Microscopy |
| Catalog No. | CCAT-HMM-0071 |
| Description | A fluorescent assay kit using JC-10, a superior cationic lipophilic dye derivative of JC-1, for measuring mitochondrial membrane potential (Delta Psi m) changes in live cells. JC-10 selectively accumulates in healthy mitochondria with high membrane potential, forming J-aggregates exhibiting red fluorescence (Ex/Em 540/590 nm). In apoptotic or metabolically compromised cells with collapsed mitochondrial membrane potential, JC-10 remains in the cytoplasm as green-fluorescent monomers (Ex/Em 490/525 nm). The kit enables ratiometric quantification of mitochondrial health, with the red-to-green fluorescence ratio serving as a direct indicator of mitochondrial polarization status. JC-10 offers improved aqueous solubility and reduced aggregation propensity compared to JC-1, providing more consistent staining and simplified protocol. The kit includes JC-10 dye (lyophilized), CCCP (carbonyl cyanide 3-chlorophenylhydrazone) positive control for mitochondrial depolarization, and 10x Assay Buffer for dilution and washing. |
| Intended Use | Quantitative measurement of mitochondrial membrane potential in live cells by flow cytometry, fluorescence microscopy, and fluorescence microplate reader. Applications: early apoptosis detection (Delta Psi m collapse precedes phosphatidylserine externalization); mitochondrial toxicity screening for drug candidates (especially hepatotoxic and cardiotoxic compounds); metabolic stress and cell health assessment; mitochondrial dysfunction studies in neurodegeneration (Parkinson's, Alzheimer's, Huntington's); ischemia-reperfusion injury models; mitochondrial uncoupling protein (UCP) and permeability transition pore (mPTP) research; cancer cell metabolism studies (Warburg effect). |
| Principle / Technology | JC-10 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide derivative) is a lipophilic cationic dye that enters mitochondria driven by the negative mitochondrial membrane potential. In healthy polarized mitochondria (Delta Psi m approximately -150 to -180 mV), JC-10 accumulates to high local concentrations, forming J-aggregates that shift the emission spectrum from green (~525 nm) to red (~590 nm). In depolarized mitochondria (Delta Psi m > -100 mV), JC-10 cannot accumulate, remaining as monomers in the cytoplasm with green fluorescence. The red/green fluorescence intensity ratio is directly proportional to Delta Psi m, allowing ratiometric quantification independent of dye loading variations, cell number, and mitochondrial mass. CCCP, a protonophore included as positive control, collapses the proton gradient across the inner mitochondrial membrane, abolishing Delta Psi m and converting all JC-10 to monomeric green form. |
| Detection Method | Prepare JC-10 working solution: dissolve lyophilized JC-10 in DMSO to 1-5 mM stock, then dilute in 1x Assay Buffer to 5-20 uM (optimize per cell type). For flow cytometry: harvest cells, wash in PBS, resuspend in JC-10 working solution at 0.5-1 x 10^6 cells/mL, incubate 15-30 min at 37 C in the dark, wash 2x with Assay Buffer, analyze by flow cytometry (FL1 channel for green monomers, 488 nm laser, 525/30 BP; FL2 channel for red J-aggregates, 488 nm laser, 585/42 BP). For microscopy: incubate adherent cells in culture medium with JC-10 working solution 15-30 min at 37 C, wash 2x, image with FITC and TRITC/Cy3 filter sets. For microplate: incubate cells in 96-well plate with JC-10, wash, read fluorescence at Ex/Em 490/525 nm (green) and 540/590 nm (red). CCCP positive control: pre-treat cells with 10-50 uM CCCP for 5-15 min before JC-10 staining. |
| Sample Type | Live mammalian cells (adherent or suspension): cell lines (HeLa, HEK293, Jurkat, HUVEC, HepG2, SH-SY5Y), primary cells (hepatocytes, cardiomyocytes, neurons, lymphocytes), isolated mitochondria. For flow cytometry: 0.5-1 x 10^6 cells per sample. For microscopy: 50-80% confluent monolayer on coverslips or chamber slides. For microplate: 10,000-50,000 cells per well (96-well). |
| Performance Range / Specifications | JC-10 fluorescence excitation: monomer 490 nm, J-aggregate 540 nm; emission: monomer 525 nm, J-aggregate 590 nm; staining time: 15-30 min at 37 C; JC-10 working concentration: 5-20 uM (optimize per cell type); CCCP positive control: 10-50 uM, 5-15 min pre-treatment; assay duration: approximately 60 min total (including staining and washing); detection limit: Delta Psi m changes as small as 5-10% detectable by flow cytometry. |
| Sensitivity / LOD | Ratiometric measurement enables detection of subtle changes in mitochondrial membrane potential (5-10% decrease) with high statistical confidence (CV <10% for triplicate measurements). JC-10 J-aggregate formation detectable at mitochondrial concentrations as low as 0.1 uM JC-10. CCCP positive control produces >10-fold decrease in red/green ratio within 5 min. |
| Specificity | JC-10 specifically accumulates in mitochondria based on membrane potential; mitochondrial localization verifiable by co-staining with MitoTracker dyes; CCCP specificity: protonophore that collapses Delta Psi m without affecting cell viability in short-term treatments (5-15 min). JC-10 does not stain lysosomes, ER, or plasma membrane at recommended concentrations. |
| Reaction Conditions / Protocol | Incubation: 15-30 min at 37 C, 5% CO2, protected from light; JC-10 working concentration: 5-20 uM in serum-free medium or 1x Assay Buffer; wash: 2x with 1x Assay Buffer or PBS; analysis: within 30 min of staining (JC-10 may slowly leak from cells). For multi-parametric analysis, combine with Annexin V-APC (no spectral overlap) or PI (after JC-10, not before). |
| Components / Formulation | JC-10 dye, 5 x 50 ug vials (lyophilized, sufficient for 500 flow cytometry tests); CCCP (10 mM in DMSO), 100 uL; 10x Assay Buffer, 50 mL; Protocol booklet. Total: 500 tests (flow cytometry; approximately 2,500 tests for microplate format). |
| Storage Conditions | Kit components at -20 C protected from light; stable for 12 months; JC-10 stock in DMSO at -20 C protected from light stable for 6 months; working solution prepare fresh and use within 2 hours; avoid repeated freeze-thaw of JC-10 DMSO stock. |
| Shelf Life | 12 months from date of manufacture at -20 C. |
| Package Specifications | 500 test kit (5 x 50 ug JC-10 vials + CCCP + Assay Buffer); also available: 100 test size (1 x 50 ug vial). |
| Product Form | JC-10: red-purple lyophilized powder; CCCP: 10 mM solution in DMSO (colorless); 10x Assay Buffer: clear liquid. JC-10 working solution: orange-red solution. |
| Quality Control | Each lot tested for: JC-10 purity >=95% (HPLC); fluorescence spectra (monomer Ex/Em 490/525 +/-5 nm, J-aggregate Ex/Em 540/590 +/-5 nm); CCCP potency: >10-fold red/green ratio decrease in Jurkat cells at 25 uM, 10 min; JC-10 mitochondrial staining pattern verified by fluorescence microscopy (colocalization with MitoTracker Deep Red FM, Pearson's r >0.85); sterility (0.1 um filtered Assay Buffer); endotoxin <0.05 EU/mL (Assay Buffer). |
| Key Features | Ratiometric red/green measurement (compensates for loading differences); superior aqueous solubility vs JC-1; CCCP positive control included; compatible with flow cytometry, microscopy, and microplate; 500 test kit size; no-wash protocol option for microscopy; minimal spectral overlap with GFP, FITC, PE in flow cytometry. |
| Purity | JC-10: >=95% by HPLC; single major peak; CCCP: >=98% purity; all buffers: molecular biology grade. |
| Concentration | JC-10 DMSO stock: 1-5 mM (prepare from 50 ug vial with appropriate DMSO volume); working: 5-20 uM in Assay Buffer; CCCP: 10 mM DMSO stock; working: 10-50 uM in medium. |
| Activity / Unit Definition | JC-10 J-aggregate formation: >5-fold fluorescence shift from green to red in polarized mitochondria; CCCP depolarization: >10-fold decrease in red/green fluorescence ratio at 25 uM. |
| Molecular Weight | JC-10: approximately 652 g/mol (iodide salt); CCCP: 204.6 g/mol. |
| Source / Origin | JC-10: synthetic carbocyanine dye; CCCP: synthetic protonophore; all reagents synthetic origin; no animal-derived components. |
| pH Range / Optimal pH | JC-10 stable at pH 6.0-8.0; optimal staining at pH 7.2-7.4; CCCP stable at pH 5.0-8.0; Assay Buffer pH 7.4. |
| Shipping Conditions | Blue ice or ambient with cold pack; JC-10 and CCCP on blue ice; Assay Buffer at ambient; kit stable for 2 weeks at ambient during shipping. |
| Expiration Date / Stability | 12 months at -20 C protected from light; JC-10 DMSO stock: 6 months at -20 C; JC-10 working solution: 2 hours at RT; do not freeze working solution. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. JC-10 is a potential irritant; use appropriate PPE. CCCP is toxic — handle with care. DMSO is a permeation enhancer — use gloves. |
| Compatibility | Flow cytometry: compatible with 488 nm laser excitation (FITC and PE channels); minimal spillover into APC, APC-Cy7, Pacific Blue channels. Compatible with Annexin V-APC, 7-AAD, PI (sequential staining), and Hoechst 33342. Compatible with fluorescence microscopy: FITC/GFP and TRITC/Cy3 filter cubes. Compatible with fluorescence microplate readers with monochromator or filter-based detection. Compatible with most mammalian cell lines and primary cells. Not compatible with: cells already expressing GFP or FITC-labeled antibodies (green channel overlap); simultaneous use with other Delta Psi m dyes (TMRE, TMRM, Rhodamine 123) may compete for mitochondrial uptake. |
| Recommended Buffer System | 1x Assay Buffer: Hank's Balanced Salt Solution (HBSS) with 20 mM HEPES, pH 7.4; serum-free formulation for optimal JC-10 loading. For adherent cell staining, JC-10 can be loaded in complete culture medium (with serum) but signal may be 20-30% lower due to serum protein binding. |
| Application Notes / Precautions | Critical: Protect JC-10 from light at all times — JC-10 is photolabile and photobleaching reduces red fluorescence signal. Centrifuge JC-10 vial before opening to collect lyophilized powder at bottom. Prepare JC-10 DMSO stock at 1-5 mM — aliquot into single-use volumes and store at -20 C. JC-10 monomer green fluorescence is partially quenched in J-aggregate form; upon depolarization, total green fluorescence increases, while red fluorescence decreases. For quantitative Delta Psi m measurement, always calculate the red/green ratio, not absolute fluorescence. CCCP positive control: pre-treat a separate aliquot of cells with 25 uM CCCP for 5-10 min at 37 C before JC-10 staining; this serves as the fully depolarized reference. For suspension cells, gentle pipetting during washing prevents cell clumping. If using FCCP as an alternative uncoupler, note that FCCP is more potent than CCCP (use 1-10 uM FCCP). Avoid using valinomycin as positive control (valinomycin collapses only the electrical component of Delta Psi m, not the total proton motive force). For time-course experiments, acquire data promptly after staining (within 30 min) as JC-10 may slowly redistribute or leak from cells. JC-10 may be a substrate for P-glycoprotein (MDR1) efflux pump — in MDR1-overexpressing cells, include cyclosporin A (2-10 uM) or verapamil (10-50 uM) in staining solution to inhibit efflux. |
| Batch-to-Batch Consistency | JC-10 HPLC purity >=95% for every lot; CCCP depolarization >10-fold red/green ratio decrease verified; mitochondrial colocalization Pearson's r >0.85; fluorescence spectra within specification. |
For research use only, not for clinical use.
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