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| Product Name | JC-1 Mitochondrial Membrane Potential Fluorometric Assay Kit |
| Catalog No. | CATR-HMM-0058 |
| Description | Fluorometric assay kit using JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide), a lipophilic cationic dye that exhibits potential-dependent accumulation in mitochondria. In healthy cells with high mitochondrial membrane potential, JC-1 forms J-aggregates emitting red fluorescence (Ex/Em 585/590 nm). In apoptotic or metabolically compromised cells with collapsed membrane potential, JC-1 remains as green-fluorescent monomers (Ex/Em 514/529 nm). The red-to-green fluorescence ratio provides a quantitative ratiometric measure of mitochondrial health that is independent of cell number, dye loading efficiency, and photobleaching artifacts. |
| Intended Use | Quantitative measurement of mitochondrial membrane potential changes in live cells during apoptosis, drug cytotoxicity screening, mitochondrial dysfunction studies, and metabolic research using fluorescence microscopy, flow cytometry, or microplate reader detection. |
| Principle / Technology | JC-1 dye accumulates in mitochondria proportional to membrane potential (negative inside). Above threshold potential (~140 mV), dye forms J-aggregates with red-shifted fluorescence. Loss of membrane potential prevents aggregation, yielding green monomer fluorescence. Ratiometric analysis normalizes for dye loading variations. |
| Detection Method | Fluorescence microscopy (FITC/TRITC channels), flow cytometry (488 nm excitation, 530/30 and 585/42 emission filters), or fluorescence microplate reader (excitation 485/535 nm, emission 530/590 nm). |
| Sample Type | Adherent and suspension cultured mammalian cells; primary cells; isolated mitochondria. |
| Performance Range / Specifications | Linear detection range: 0.1-10 µM JC-1 working concentration; distinguishable membrane potential changes from -220 mV to -50 mV; applicable to 1×10^4 to 1×10^7 cells per condition. |
| Sensitivity / LOD | Detectable mitochondrial depolarization induced by as little as 1 µM CCCP (carbonyl cyanide m-chlorophenyl hydrazone) within 15 minutes; resolution of 10-15 mV membrane potential change. |
| Specificity | JC-1 selectively accumulates in polarized mitochondria based on membrane potential; does not stain plasma membrane, endoplasmic reticulum, or lysosomes at recommended concentrations. |
| Reaction Conditions / Protocol | Incubate cells with 2 µM JC-1 in culture medium or assay buffer for 15-30 minutes at 37 °C; wash twice with assay buffer; analyze immediately. |
| Components / Formulation | JC-1 dye (lyophilized, 5 mg), DMSO (1 mL, anhydrous), CCCP positive control (10 mM in DMSO, 50 µL), 10× Assay Buffer (50 mL), detailed protocol. |
| Storage Conditions | Store at -20 °C protected from light; JC-1 stock solution stable for 1 month at -20 °C in DMSO. |
| Shelf Life | 12 months from date of manufacture. |
| Package Specifications | 100 tests, 500 tests, 1000 tests (based on 500 µL staining volume per test). |
| Product Form | Lyophilized dye powder; DMSO for reconstitution; concentrated buffer solution. |
| Quality Control | Each lot tested for JC-1 aggregate/monomer fluorescence ratio in healthy HeLa cells vs. CCCP-treated cells; verified ratio change >5-fold between conditions. |
| Key Features | Ratiometric measurement eliminates cell-number artifacts; dual-color detection; compatible with microscopy, flow cytometry, and plate reader; CCCP positive control included; suitable for 96-well and 384-well plate formats. |
| Purity | JC-1 dye purity ≥95% by HPLC; DMSO anhydrous <0.005% water. |
| Concentration | JC-1 stock: 5 mg/mL in DMSO (prepare before use). |
| Activity / Unit Definition | JC-1 fluorescence quantum yield: monomers 0.18, J-aggregates 0.12 in aqueous buffer. |
| Molecular Weight | JC-1: 652.23 g/mol (C25H27Cl4IN4). |
| Source / Origin | Synthetic carbocyanine dye manufactured under cGMP conditions. |
| pH Range / Optimal pH | Optimal staining at pH 7.4; buffer pH range 7.2-7.8 compatible. |
| Shipping Conditions | Ambient or cold pack; JC-1 dye is light-sensitive; protect during transit. |
| Expiration Date / Stability | 12 months at -20 °C; reconstituted JC-1 solution stable for 1 month at -20 °C in anhydrous DMSO. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic applications. |
| Compatibility | Compatible with standard cell culture media (DMEM, RPMI-1640, MEM). Avoid media with phenol red for fluorescence readings — use phenol red-free medium. Compatible with GFP-expressing cells using sequential imaging (green before red). |
| Recommended Buffer System | 10× Assay Buffer: HEPES 200 mM, pH 7.4; dilute to 1× with deionized water. |
| Application Notes / Precautions | Avoid repeated freeze-thaw of JC-1 stock solution. Pre-warm buffer to 37 °C for optimal staining. Gate out debris by forward/side scatter in flow cytometry. Use CCCP at 10-50 µM for 5-15 minutes as positive control. JC-1 is sensitive to light — work under subdued lighting. For 384-well plate format, use 20 µL staining volume at 5 µM JC-1. |
| Batch-to-Batch Consistency | JC-1 dye content within ±5% of label claim; aggregate-to-monomer ratio in control cells within ±15% of reference lot. |
For research use only, not for clinical use.
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