| Product Name | Live Cell Caspase-3 Activity and Annexin V Apoptosis Detection Reagent |
| Catalog No. | CCAT-HMM-0031 |
| Description | GreenNuc Caspase-3 is a peptide DEVD coupled with a DNA green fluorescent dye. The DEVD sequence in this substrate is the recognition sequence for caspase 3/7 and carries a large negative charge. These negative charges repel the negative charges on DNA, preventing the DNA green fluorescent dye in the substrate from binding to DNA and thus from being excited to emit green fluorescence. The initially non-fluorescent substrate crosses the cell membrane into the cytoplasm, where it is recognized and cleaved by caspase-3/7 in apoptotic cells, releasing activated DNA green fluorescent dye molecules. These activated DNA green fluorescent dye molecules migrate into the nucleus, bind to DNA, and form GreenNuc-DNA complexes, which can then be excited to produce bright green fluorescence. In normal living cells, phosphatidylserine (PS) is located on the cytoplasmic side of the cell membrane. In apoptotic cells, PS is internalized from the inner side of the plasma membrane to the cell surface (the outer side of the cell membrane), thereby exposing PS to the extracellular environment. During the apoptosis of white blood cells, the PS markers on the surface of white blood cells can be recognized by macrophages, ultimately leading to their phagocytosis by macrophages. Human vascular anticoagulant Annexin V is a Ca²⁺-dependent phospholipid-binding protein with a molecular weight of 35–36 kDa and high affinity for PS. By staining cells with Annexin V labeled with red fluorescent mCherry (Annexin V-mCherry), the important apoptotic feature of PS inversion can be detected very simply and directly using fluorescence detection equipment such as fluorescence microscopes, laser confocal microscopes, or flow cytometers. It should be noted that in cells undergoing necrosis, due to the disruption of cell membrane integrity, PS located on the inner side of the cell membrane is also stained by Annexin V-mCherry. |
| Application | This product uses a novel cell membrane-permeable Caspase-3/7 green fluorescent substrate, GreenNuc Caspase-3 Substrate, combined with the red fluorescent apoptosis probe Annexin V-mCherry to detect Caspase-3 activity and apoptosis in cultured cells. |
| Applicable Instruments | Flow cytometer, fluorescence microscope, or other fluorescence detection equipment |
| Materials to Bring | 1 × PBS, pH 7.2-7.6; sterile ddH₂O |
| Storage | Store at -20°C away from light. |
| Shelf Life | 1 year |
| Notes | Avoid microbial contamination, as bacterial or fungal contamination can severely affect the detection results. After staining, testing should be performed as soon as possible, as prolonged exposure may lead to an increase in the number of apoptotic or necrotic cells. If trypsin was used during cell collection, care must be taken to remove any residual trypsin. Residual trypsin can digest and degrade Annexin V-mCherry, ultimately resulting in staining failure. GreenNuc Caspase-3 Substrate is not very stable and should be avoided from repeated freeze-thaw cycles (generally not exceeding three times). When using flow cytometry, if the Annexin V-mCherry or GreenNuc signal is too strong and cannot be improved by adjusting relevant settings and parameters, dilute Annexin V-mCherry or GreenNuc with PBS by 3–10 times before testing. Fluorescent substances are prone to quenching. When performing fluorescence observations, minimize observation time and ensure light-protected storage during handling and storage. For your safety and health, please wear a lab coat and disposable gloves during handling. |
For research use only, not for clinical use.
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