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| Product Name | JC-1 Mitochondrial Membrane Potential Assay Kit |
| Catalog No. | RESK-0195 |
| Description | The JC-1 Mitochondrial Membrane Potential Assay Kit uses JC-1 as a fluorescent probe to rapidly and sensitively detect changes in the mitochondrial membrane potential of cells, tissues or purified mitochondria. It can be used for early apoptosis detection and is a commonly used method for this purpose. JC-1 is an ideal fluorescent probe widely used to detect mitochondrial membrane potential (delta psi m); the dye accumulates in mitochondria in a potential-dependent manner. In healthy mitochondria, JC-1 aggregates in the mitochondrial matrix to form J-aggregates that emit intense red fluorescence (Ex = 585 nm, Em = 590 nm). In unhealthy mitochondria, where the membrane potential is decreased or lost, JC-1 exists only as monomers in the cytosol, producing green fluorescence (Ex = 514 nm, Em = 529 nm). JC-1 supports both qualitative detection (the color change directly reflects membrane potential changes) and quantitative detection (the degree of mitochondrial depolarization is measured by the red/green fluorescence intensity ratio). Only conventional red and green fluorescence observation settings are required. Disruption of the mitochondrial membrane potential is a hallmark event in the early stage of apoptosis. Apoptotic induction alters membrane permeability, causing mitochondrial proteins such as cytochrome c, Smac/DIABLO, HtrA2/OMI, endonuclease G (EndoG) and apoptosis-inducing factor (AIF) to be released from the mitochondrial matrix into the cytosol. Cytochrome c release accompanies complete loss of membrane potential and triggers the caspase cascade of apoptosis. The kit provides CCCP as a positive control for inducing a decrease in mitochondrial membrane potential. It is sufficient for 100 samples in 6-well plates or 200 samples in 12-well plates. |
| Intended Use | Rapid, sensitive detection of mitochondrial membrane potential changes in cells, tissues or purified mitochondria; detection of early-stage apoptosis. |
| Principle / Technology | JC-1 accumulates in mitochondria in a potential-dependent manner. At high membrane potential it forms red-fluorescent J-aggregates in the matrix (Ex = 585 nm, Em = 590 nm); upon depolarization it remains as green-fluorescent monomers in the cytosol (Ex = 514 nm, Em = 529 nm). The red/green fluorescence ratio quantifies depolarization. Loss of mitochondrial membrane potential is an early apoptotic event linked to the release of cytochrome c and other mitochondrial proteins and subsequent caspase cascade activation. |
| Detection Method | Fluorescence detection using conventional red and green fluorescence settings (J-aggregate: Ex = 585 nm, Em = 590 nm; monomer: Ex = 514 nm, Em = 529 nm); qualitative assessment by color change and quantitative assessment by the red/green fluorescence intensity ratio. Suitable for fluorescence microscopy and flow cytometry. |
| Sample Type | Living cells (adherent or suspension; digest adherent cells into a cell suspension before staining for flow cytometry), tissues processed into single-cell suspensions, or freshly purified intact mitochondria. Samples must be viable/live; paraffin sections and frozen sections are not suitable. |
| Performance Range / Specifications | Sufficient for 100 samples in 6-well plates or 200 samples in 12-well plates. CCCP is provided as a positive control for inducing mitochondrial membrane potential decrease. |
| Reaction Conditions / Protocol | Preparation of JC-1 working solution: dilute JC-1 (200x) with ultrapure water at a ratio of 50 uL JC-1 (200x) per 8 mL ultrapure water, vortex vigorously to fully dissolve and mix, then add 2 mL JC-1 Staining Buffer (5x). If green insoluble precipitate remains, add Tween 20 to 0.1% (v/v). For purified intact mitochondria: add 0.1 mL of purified mitochondria containing 10-100 μg total protein to 0.9 mL of JC-1 staining working solution (1x); detection can be performed 3-5 minutes after adding the JC-1 working solution. Complete detection within 30 minutes after staining. |
| Components / Formulation | JC-1 (200x), JC-1 Staining Buffer (5x), ultrapure water, and CCCP (positive control for inducing mitochondrial membrane potential decrease). The JC-1 probe is supplied as a liquid in this kit. |
| Storage Conditions | Store at -20 °C protected from light; avoid repeated freeze-thaw cycles. Ultrapure water and JC-1 Staining Buffer (5x) may also be stored at 4 °C. |
| Shelf Life | 1 year at -20 °C, protected from light. |
| Package Specifications | 100 T |
| Quality Control | CCCP positive control is included to validate induction of mitochondrial membrane potential decrease in each assay. |
| Shipping Conditions | Shipped on wet ice. |
| Regulatory / Compliance | For research use only. |
| Recommended Buffer System | JC-1 Staining Buffer (5x), supplied in the kit, diluted to 1x with ultrapure water. |
| Application Notes / Precautions | Samples must be live cells; paraffin sections and frozen sections cannot be used. For tissue samples, prepare a single-cell suspension first and follow the suspension-cell procedure (the preparation protocol should be optimized empirically). For flow cytometry of adherent cells, digest the cells into a suspension first, then stain. Do not fix cells after staining if detection cannot be performed immediately: JC-1 works only with live cells, and fluorescence quenches with prolonged standing; complete detection within 30 minutes after staining. If the negative control shows green fluorescence, the probe concentration may be too high; reduce the probe concentration appropriately. Green monomers are dispersed in the cytosol while red J-aggregates bind to mitochondria, so the visible cell morphology also depends on mitochondrial distribution. For purified mitochondria, use 0.1 mL of mitochondria (10-100 μg total protein) per 0.9 mL of 1x working solution and detect 3-5 minutes after adding JC-1; frozen-thawed purified mitochondria cannot be used because freeze-thawing disrupts the mitochondrial membrane. If green precipitate appears during working solution preparation, vortex vigorously after diluting JC-1 (200x) in ultrapure water, then add the 5x staining buffer; add 0.1% Tween 20 if insoluble precipitate persists. |
For research use only, not for clinical use.
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