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| Product Name | Annexin V-APC Apoptosis Detection Kit for Flow Cytometry |
| Catalog No. | CCAT-HMM-0062 |
| Description | An APC-conjugated Annexin V apoptosis detection kit for flow cytometric discrimination of early apoptotic, late apoptotic, and necrotic cells. Annexin V-APC binds with high affinity to phosphatidylserine (PS) translocated to the outer leaflet of the plasma membrane during early apoptosis. Using the far-red APC fluorophore (Ex/Em 650/660 nm) enables multiplex apoptosis detection with GFP, FITC, and PE-labeled markers in the same sample, expanding multicolor panel design options. |
| Intended Use | Detection and quantification of early and late apoptotic cells by flow cytometry; discrimination of apoptosis from necrosis; evaluation of apoptosis-inducing therapeutic agents; cell health assessment in immunophenotyping panels; quality control of cell therapy products. |
| Principle / Technology | Recombinant Annexin V conjugated to allophycocyanin (APC) binds with high affinity (Kd ~0.1 nM) to phosphatidylserine exposed on the outer leaflet of apoptotic cell membranes in a calcium-dependent manner; co-staining with a viability dye (7-AAD or PI) distinguishes early apoptotic (Annexin V+/viability dye-) from late apoptotic/necrotic (double positive) cells |
| Detection Method | Flow cytometry; APC excitation 633–640 nm (red laser), emission 660/20 nm bandpass filter; 7-AAD or PI emission in PerCP or PE-Texas Red channel |
| Sample Type | Single cell suspension from cultured cells, dissociated tissue, blood, or bone marrow; 1 × 10^5 to 1 × 10^6 cells per test |
| Sensitivity / LOD | Detects apoptosis in populations as low as 1% of total cells; resolves early apoptotic cells before membrane integrity loss; Annexin V-APC signal-to-noise ratio >50:1 on positive control cells |
| Specificity | Calcium-dependent, high-affinity binding to phosphatidylserine; minimal binding to phosphatidylcholine or phosphatidylethanolamine; requires calcium (2.5 mM) in binding buffer for optimal binding |
| Reaction Conditions / Protocol | Harvest cells (include floating cells for adherent cultures); wash twice with cold PBS; resuspend in 100 µL 1× binding buffer at 1 × 10^6 cells/mL; add 5 µL Annexin V-APC and 5 µL 7-AAD viability dye; incubate 15 minutes at room temperature in dark; add 400 µL binding buffer; analyze immediately by flow cytometry |
| Components / Formulation | Annexin V-APC conjugate (200 µg/mL in PBS with 0.1% BSA), 7-AAD viability staining solution (1 mg/mL), binding buffer (10× concentrate, contains HEPES, NaCl, CaCl2), positive control apoptosis inducer (camptothecin, 2 mM in DMSO) |
| Storage Conditions | Store at 2–8 °C; protect from light; do not freeze |
| Shelf Life | 12 months from manufacture date; use within 6 months after opening |
| Package Specifications | 50 tests, 100 tests, 200 tests; test defined as 1 × 10^6 cells per 100 µL staining volume |
| Product Form | Liquid reagents, ready-to-use; 10× binding buffer diluted to 1× with deionized water before use |
| Key Features | APC conjugation enables multiplexing with FITC, PE, and PerCP conjugates; bright far-red signal with minimal spectral overlap; recombinant Annexin V ensures lot-to-lot consistency; includes positive control inducer for assay validation; 7-AAD included for viability discrimination; calcium-optimized binding buffer maximizes PS binding |
| Purity | Recombinant Annexin V-APC purity >95% by SDS-PAGE; F/P ratio 1.5–2.5 mol APC per mol protein; endotoxin <0.1 EU/µg |
| Concentration | As specified per kit; sufficient for stated number of tests |
| Activity / Unit Definition | Apoptosis induction confirmed by positive control treatment producing ≥5-fold signal increase |
| Molecular Weight | Varies by dye and enzyme component |
| Source / Origin | Recombinant annexin proteins; synthetic fluorophore conjugates; chromogenic/fluorogenic peptide substrates |
| pH Range / Optimal pH | Binding/washing buffer pH 7.2–7.4 |
| Shipping Conditions | Cold pack 2–8 °C; protect from light; do not freeze which denatures protein conjugate |
| Expiration Date / Stability | 12 months at 2–8 °C protected from light; 6 months after opening; discard if solution becomes turbid or develops precipitate; binding buffer diluted to 1× stable for 1 month at 4 °C |
| Regulatory / Compliance | For laboratory and research use only; RUO; manufactured under ISO 9001; APC is a phycobiliprotein — non-toxic; 7-AAD is a potential mutagen — handle with gloves |
| Compatibility | Compatible with all flow cytometers equipped with red (633–640 nm) laser; use polypropylene or siliconized tubes to minimize cell adherence; calcium-containing binding buffer may cause cell clumping in some cell types — reduce Ca2+ to 1 mM if clumping occurs |
| Recommended Buffer System | Annexin binding buffer with calcium; cell lysis buffer with protease inhibitors for caspase assays |
| Application Notes / Precautions | Always include unstained control, Annexin V-APC single stain, and 7-AAD single stain for compensation setup. For camptothecin positive control, treat Jurkat cells with 2–5 µM camptothecin for 4–6 hours at 37 °C. Do not fix cells after staining — fixation may increase non-specific Annexin V binding and alter scatter properties. Analyze within 1 hour of staining for optimal results. |
| Batch-to-Batch Consistency | Positive control signal within ±20% of reference lot; staining pattern consistent |
For research use only, not for clinical use.
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