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| Product Name | Mitochondrial Permeability Transition Pore Assay Kit (Flow Cytometry) |
| Catalog No. | RESK-0199 |
| Description | The mitochondrial permeability transition pore (mPTP), also called the mitochondrial megachannel, is a non-selective, high-conductance channel spanning the inner and outer mitochondrial membranes and composed of a multiprotein complex. The mPTP may participate in the release of mitochondrial components during cell death. In normal cells, the inner mitochondrial membrane maintains the mitochondrial potential gradient required for respiration and energy supply, and a low-conductance permeability transition pore switches between open and closed states as Ca2+ is taken up and released. During apoptosis and pathological cell death, the permeability of the pore changes: Ca2+ overload, oxidation of mitochondrial glutathione, elevated reactive oxygen species, and subsequent events such as cytochrome c release and loss of mitochondrial membrane potential all lead to mPTP activation. This kit provides a more direct method for detecting mPTP opening than assays based solely on mitochondrial membrane potential. Cells are first loaded by passive transport with Calcein AM, a cell-staining reagent for fluorescent labeling of live cells that readily penetrates the cell membrane and is cleaved by intracellular esterases into membrane-impermeant polar calcein, which is retained in the cell and produces strong green fluorescence in the cytosol, including the mitochondria. Upon addition of CoCl2, the cytosolic fluorescence is quenched, leaving only the mitochondrial fluorescence. As a control, cells loaded with Calcein AM and CoCl2 are treated with ionomycin to increase Ca2+ loading, which activates the mPTP and quenches the mitochondrial fluorescence. Based on a 1 mL labeling system, the kit is sufficient for 100 assays. Maximum excitation wavelength: 494 nm; maximum emission wavelength: 517 nm. |
| Intended Use | For direct detection of mitochondrial permeability transition pore (mPTP) opening in cells by flow cytometry. |
| Principle / Technology | Calcein AM passively enters live cells and is cleaved by intracellular esterases into membrane-impermeant calcein, yielding strong green fluorescence in both cytosol and mitochondria. CoCl2 quenches the cytosolic fluorescence, leaving only mitochondrial fluorescence. When the mPTP opens (e.g., upon ionomycin-induced Ca2+ overload in the control), cobalt enters the mitochondria and quenches the mitochondrial fluorescence as well; thus, loss of mitochondrial calcein fluorescence indicates mPTP opening. |
| Detection Method | Flow cytometry; maximum excitation 494 nm, maximum emission 517 nm. |
| Sample Type | Live cells |
| Performance Range / Specifications | Directly detects mPTP opening. Sufficient for 100 assays based on a 1 mL labeling system. Ex/Em: 494/517 nm (maximum). |
| Reaction Conditions / Protocol | Load cells with Calcein AM, then add CoCl2 to quench cytosolic fluorescence, leaving only mitochondrial fluorescence. As a control, treat cells loaded with Calcein AM and CoCl2 with ionomycin to increase Ca2+ loading and activate the mPTP, quenching mitochondrial fluorescence. Analyze by flow cytometry. |
| Storage Conditions | Store at -20 °C, protected from light and dry; avoid repeated freeze-thaw cycles. |
| Shelf Life | At least 6 months |
| Package Specifications | 100 T |
| Product Form | Kit |
| Shipping Conditions | Shipped on ice packs |
| Expiration Date / Stability | Valid for at least 6 months when stored at -20 °C protected from light; avoid repeated freeze-thaw cycles. |
| Regulatory / Compliance | For research use only. |
| Compatibility | Flow cytometry analysis |
| Application Notes / Precautions | Protect from light. Avoid repeated freeze-thaw cycles. Use the ionomycin-treated control to confirm mPTP-dependent quenching of mitochondrial fluorescence. |
For research use only, not for clinical use.
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