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| Product Name | Annexin V-Fluor 647/PI Apoptosis Detection Kit |
| Catalog No. | RESK-0142 |
| Description | This kit uses Annexin V labeled with Fluor 647, a far-red fluorescent dye (Ex/Em approximately 650/665 nm), as a probe to detect the onset of early apoptosis. Detection can be performed by flow cytometry or other fluorescence-based instruments. In viable cells, phosphatidylserine (PS) is localized on the inner leaflet of the plasma membrane. During early apoptosis, PS is translocated from the inner to the outer leaflet and becomes exposed to the extracellular environment. Annexin V is a Ca2+-dependent phospholipid-binding protein with a molecular weight of 35-36 kDa that binds PS with high affinity, allowing fluorescently labeled Annexin V to bind to the membrane of early apoptotic cells via the exposed PS. Propidium iodide (PI) is a nucleic acid dye that cannot penetrate the intact membrane of viable or early apoptotic cells, but can enter late apoptotic and necrotic cells and stain the nucleus red. When Annexin V and PI are used in combination, PI is excluded from viable cells (Annexin V-/PI-) and early apoptotic cells (Annexin V+/PI-), while late apoptotic and necrotic cells are co-stained and appear double positive (Annexin V+/PI+). This kit requires flow cytometry for detection. |
| Intended Use | For flow cytometric detection and discrimination of viable, early apoptotic, late apoptotic and necrotic cells. For research use only. |
| Principle / Technology | In viable cells, phosphatidylserine (PS) is localized on the inner leaflet of the plasma membrane. During early apoptosis, PS is translocated from the inner to the outer leaflet and becomes exposed to the extracellular environment. Annexin V is a Ca2+-dependent phospholipid-binding protein with a molecular weight of 35-36 kDa that binds PS with high affinity, allowing fluorescently labeled Annexin V to bind to the membrane of early apoptotic cells via the exposed PS. Propidium iodide (PI) is a nucleic acid dye that cannot penetrate the intact membrane of viable or early apoptotic cells, but can enter late apoptotic and necrotic cells and stain the nucleus red. When Annexin V and PI are used in combination, PI is excluded from viable cells (Annexin V-/PI-) and early apoptotic cells (Annexin V+/PI-), while late apoptotic and necrotic cells are co-stained and appear double positive (Annexin V+/PI+). Annexin V is conjugated to the far-red dye Fluor 647 (Ex/Em approximately 650/665 nm). |
| Detection Method | Flow cytometry (required); signal may also be visualized with fluorescence microplate readers, fluorescence or confocal microscopes where applicable |
| Sample Type | Cultured cells (adherent or suspension) |
| Performance Range / Specifications | Convenient and accurate detection |
| Specificity | Annexin V binds externalized phosphatidylserine in a Ca2+-dependent manner with high affinity; PI is excluded by intact membranes and stains only late apoptotic/necrotic cells |
| Reaction Conditions / Protocol | Stain cells with Annexin V-Fluor 647 and PI in the kit Binding Buffer according to the manual. Perform observation immediately after staining; the entire observation should be completed within 30 min. If the kit provides a dilution buffer, use it to prepare the staining working solution; otherwise HBSS, serum-free medium or PBS may be used. For coverslip (cell climbing slice) observation, add enough working solution to submerge the cells; for flow cytometry, follow the dosage recommended in the manual. |
| Components / Formulation | Annexin V-Fluor 647 conjugate, 0.4 mg/mL Propidium Iodide (PI), 20 ug/mL Binding Buffer |
| Storage Conditions | Store at -20°C protected from light; avoid repeated freeze-thaw cycles. For repeated use within a short period, store at 4°C protected from light. |
| Shelf Life | 1 year when stored at -20°C protected from light; 6 months when stored at 4°C protected from light |
| Package Specifications | 20 T 50 T 100 T |
| Product Form | Liquid kit (ready-to-use fluorescent probes and buffer) |
| Concentration | Annexin V-Fluor 647: 0.4 mg/mL; PI: 20 ug/mL |
| Molecular Weight | Annexin V: 35-36 kDa |
| Shipping Conditions | Shipped on wet ice |
| Expiration Date / Stability | Stable for 1 year at -20°C protected from light (avoid repeated freeze-thaw); stable for 6 months at 4°C protected from light |
| Regulatory / Compliance | For research use only |
| Compatibility | Flow cytometers equipped with a far-red/APC-type detection channel (Ex/Em approximately 650/665 nm) and a PI channel |
| Recommended Buffer System | Kit Binding Buffer; HBSS, serum-free medium or PBS may be used if no dilution buffer is supplied |
| Application Notes / Precautions | Observe samples immediately after staining and complete the observation within 30 min. Protect the fluorescent probe from light during operation and incubation to avoid quenching. If the fluorescence signal is weak, moderately prolong incubation time or increase working solution concentration, and use cells in good condition. Rapid fluorescence quenching may result from excessive reagent concentration; reduce the concentration, use low-energy excitation while searching for fields, or add an anti-fade reagent. Annexin V staining cannot be performed on fixed cells. |
For research use only, not for clinical use.
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