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| Product Name | Annexin V-FITC and 7-AAD Apoptosis Detection Kit, Dual Staining |
| Catalog No. | CATR-HMM-0063 |
| Description | Dual-fluorescence apoptosis detection kit combining Annexin V conjugated to fluorescein isothiocyanate (FITC) and 7-aminoactinomycin D (7-AAD) for simultaneous differentiation of viable, early apoptotic, late apoptotic, and necrotic cell populations by flow cytometry. In early apoptosis, phosphatidylserine (PS) translocates from the inner to outer plasma membrane leaflet where it is bound by FITC-Annexin V in a calcium-dependent manner. 7-AAD, a membrane-impermeant DNA intercalating dye, enters only late apoptotic and necrotic cells with compromised membrane integrity. The combination discriminates: viable (FITC-/7-AAD-), early apoptotic (FITC+/7-AAD-), late apoptotic (FITC+/7-AAD+), and necrotic (FITC-/7-AAD+). |
| Intended Use | Quantitative detection and discrimination of apoptotic and necrotic cell populations in suspension and adherent cell cultures by flow cytometry for drug mechanism-of-action studies, toxicology screening, and basic apoptosis research. |
| Principle / Technology | Annexin V binds exposed phosphatidylserine with high affinity (Kd ~0.1 nM) in Ca2+-dependent manner; 7-AAD intercalates between GC-rich DNA regions only when plasma membrane integrity is compromised; dual staining enables four-quadrant population discrimination. |
| Detection Method | Flow cytometry: 488 nm excitation; FITC detection 530/30 nm (FL1); 7-AAD detection >650 nm longpass or 670/14 nm (FL3/FL4); compensation required between FITC and 7-AAD channels. |
| Sample Type | Freshly harvested cultured cells (adherent and suspension); primary lymphocytes, thymocytes, bone marrow cells; not recommended for fixed or permeabilized cells. |
| Performance Range / Specifications | Quantifiable apoptosis range: 1-99% of cell population; staining resolution allows discrimination of populations differing by 5% or more in apoptosis rate. |
| Sensitivity / LOD | Detection of as few as 0.5% early apoptotic cells in a population of 10,000 events; Annexin V binding detectable at PS surface density of <10,000 molecules per cell. |
| Specificity | Recombinant Annexin V binds PS with >1000-fold selectivity over phosphatidylcholine and phosphatidylethanolamine; 7-AAD selectively stains dsDNA in membrane-compromised cells; does not enter intact cells within 15-minute staining. |
| Reaction Conditions / Protocol | Harvest cells; wash with cold PBS; resuspend in Binding Buffer (1×10^5-1×10^6 cells/100 µL); add 5 µL Annexin V-FITC and 5 µL 7-AAD; incubate 15 min at RT in dark; add 400 µL Binding Buffer; analyze within 1 hour by flow cytometry. |
| Components / Formulation | Annexin V-FITC conjugate (100 tests), 7-AAD viability dye (100 tests), 10× Annexin V Binding Buffer (25 mL), detailed protocol. |
| Storage Conditions | Store at 2-8 °C protected from light; do not freeze. |
| Shelf Life | 12 months from date of manufacture. |
| Package Specifications | 100 tests, 200 tests, 500 tests (100 µL staining volume per test). |
| Product Form | Liquid reagents: FITC-Annexin V in Tris-buffered saline with BSA; 7-AAD in DMSO solution; 10× binding buffer concentrate. |
| Quality Control | Each lot tested by flow cytometry using camptothecin-treated and untreated Jurkat cells; verified four-quadrant population distribution; Annexin V binding verified using Ca2+-chelated negative control. |
| Key Features | Dual-parameter apoptosis detection; discriminates four cell populations; recombinant Annexin V for lot-to-lot consistency; ready-to-use format; no fixation required; compatible with most flow cytometers. |
| Purity | Recombinant Annexin V ≥95% by SDS-PAGE; FITC labeling ratio 2-4 moles FITC per mole protein; 7-AAD ≥95% by HPLC. |
| Concentration | Annexin V-FITC: 0.5 µg/test (5 µL per 100 µL staining volume). |
| Activity / Unit Definition | Annexin V PS binding activity validated by calcium-dependent erythrocyte binding assay; Kd for PS ~0.1 nM. |
| Molecular Weight | Annexin V: ~35.8 kDa; FITC: 389.38 g/mol; 7-AAD: 1270.43 g/mol (C62H87N13O16). |
| Source / Origin | Recombinant human Annexin V expressed in E. coli; synthetic FITC and 7-AAD. |
| pH Range / Optimal pH | 1× Binding Buffer pH 7.4 ± 0.1; Ca2+ concentration 2.5 mM. |
| Shipping Conditions | Cold pack (2-8 °C); protect from light. |
| Expiration Date / Stability | 12 months at 2-8 °C; after opening, use within 6 months and store protected from light. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic applications. Contains DMSO in 7-AAD solution — handle according to institutional chemical safety procedures. |
| Compatibility | Compatible with standard flow cytometers equipped with 488 nm laser (FL1/FL2/FL3 detectors). Verify compensation settings using single-stained controls. Compatible with most mammalian cell types including human, mouse, rat, and non-human primate cells. |
| Recommended Buffer System | 10× Binding Buffer: HEPES 100 mM, NaCl 1.4 M, CaCl2 25 mM, pH 7.4. |
| Application Notes / Precautions | Prepare single-stained compensation controls: unstained cells, Annexin V-FITC only, and 7-AAD only (use heat-killed cells at 65 °C for 5 min for 7-AAD positive). Avoid prolonged incubation beyond 30 minutes as Annexin V may internalize. Over-trypsinization during cell harvesting damages membrane PS asymmetry — use gentle dissociation methods. EDTA in trypsin inhibits Ca2+-dependent Annexin V binding; wash cells thoroughly after trypsinization. For adherent cells, include floating cells in analysis as they often represent apoptotic populations. |
| Batch-to-Batch Consistency | Annexin V-FITC protein concentration and fluorochrome labeling ratio within ±15% of reference lot; 7-AAD staining intensity within ±20%. |
For research use only, not for clinical use.
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