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| Product Name | Annexin V-PE/7-AAD Apoptosis Detection Kit |
| Catalog No. | RESK-0144 |
| Description | This kit uses Annexin V labeled with the red fluorescent dye PE (phycoerythrin) as a probe to detect the onset of early apoptosis. Detection can be performed by fluorescence microscopy, 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. 7-AAD (7-aminoactinomycin D) is a nucleic acid dye with fluorescence properties similar to PI but with a narrower emission spectrum, causing less interference with other detection channels; it is an optimal substitute for PI in multicolor fluorescence analysis and can be used in combination with Annexin V. The dye cannot penetrate the intact membrane of viable or early apoptotic cells, but can enter late apoptotic or necrotic cells and bind their DNA. When Annexin V-PE and 7-AAD are used in combination, 7-AAD is excluded from viable cells (Annexin V-/7-AAD-) and early apoptotic cells (Annexin V+/7-AAD-), while late apoptotic and necrotic cells are co-stained and appear double positive (Annexin V+/7-AAD+). |
| Intended Use | For the detection and discrimination of viable, early apoptotic, late apoptotic and necrotic cells by fluorescence microscopy, flow cytometry or other fluorescence detection instruments. 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. 7-AAD (7-aminoactinomycin D) is a nucleic acid dye with fluorescence properties similar to PI but with a narrower emission spectrum, causing less interference with other detection channels; it is an optimal substitute for PI in multicolor fluorescence analysis and can be used in combination with Annexin V. The dye cannot penetrate the intact membrane of viable or early apoptotic cells, but can enter late apoptotic or necrotic cells and bind their DNA. When Annexin V-PE and 7-AAD are used in combination, 7-AAD is excluded from viable cells (Annexin V-/7-AAD-) and early apoptotic cells (Annexin V+/7-AAD-), while late apoptotic and necrotic cells are co-stained and appear double positive (Annexin V+/7-AAD+). |
| Detection Method | Flow cytometry, fluorescence microscopy or other fluorescence detection instruments. Annexin V-PE: Ex = 488 nm, Em = 578 nm (orange-red fluorescence); FL2 channel recommended. 7-AAD: Ex = 546 nm, Em = 647 nm (red fluorescence); FL3 channel recommended. |
| 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; 7-AAD is excluded by intact membranes and stains only late apoptotic/necrotic cells |
| Reaction Conditions / Protocol | Stain cells with Annexin V-PE and 7-AAD in 1x Binding Buffer (containing 2.5 mM Ca2+) according to the kit manual, then analyze by flow cytometry or fluorescence microscopy. Viable cells (Annexin V-/7-AAD-); early apoptotic cells (Annexin V+/7-AAD-); late apoptotic/necrotic cells (Annexin V+/7-AAD+). |
| Components / Formulation | Annexin V-PE conjugate 7-AAD Binding Buffer (containing 2.5 mM Ca2+) |
| Storage Conditions | Store all components at 4°C; do not freeze. Annexin V-PE and 7-AAD must be protected from light. |
| Shelf Life | 1 year 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) |
| Molecular Weight | Annexin V: 35-36 kDa |
| Shipping Conditions | Shipped on wet ice |
| Expiration Date / Stability | Stable for 1 year at 4°C protected from light; do not freeze |
| Regulatory / Compliance | For research use only |
| Compatibility | Flow cytometers with FL2 (PE) and FL3 (7-AAD) channels; fluorescence microscopes and other fluorescence detection instruments |
| Recommended Buffer System | 1x Binding Buffer supplied in the kit (contains 2.5 mM Ca2+) |
| Application Notes / Precautions | Annexin V binding to PS is Ca2+-dependent; the Binding Buffer contains 2.5 mM Ca2+. Use EDTA-free trypsin for adherent cell detachment; if EDTA-containing trypsin is used, wash thoroughly to remove EDTA. Remove residual PBS after washing, as phosphate can form calcium phosphate precipitates; keep the Binding Buffer cap tightly closed to prevent CO2 absorption and calcium carbonate formation, and avoid touching other buffers with the pipette tip, all of which reduce free Ca2+ and may cause assay failure. Some cell types with low membrane PS density stain poorly; consider switching cell lines or using a TUNEL-based apoptosis kit. For adherent cells, also collect floating cells after drug induction, as they are often apoptotic; discarding them lowers the measured positive rate. High double-positive background in uninduced controls usually reflects low cell viability or harsh handling: verify viability by trypan blue exclusion (>95% viable in the negative control), passage freshly recovered cells 2-3 times before use, avoid vigorous pipetting and over-digestion, and avoid induction times longer than 48 h (early apoptosis generally appears within a few hours; prolonged induction depletes nutrients and increases false positives). Confirm that the inducer used actually triggers apoptosis by including a positive drug control. |
For research use only, not for clinical use.
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