Background information
Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS). Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution across the phospholipid bilayer and is translocated to the extracellular membrane leaflet marking cells as targets of phagocytosis. Once on the outer surface of the membrane, PS can be detected by fluorescently labeled
The fluorescent dye Hoechst 33342 can slightly penetrate the membranes of normal cells, resulting in low blue staining. In contrast, the membrane permeability of apoptotic cells is increased, allowing more Hoechst 33342 to enter than in normal cells, thus producing a higher fluorescence intensity. In addition, the structural changes in chromosomal DNA of apoptotic cells enable the dye to bind more effectively to DNA. Furthermore, the function of the P-glycoprotein pump on the membrane of apoptotic cells is impaired, reducing its ability to efflux Hoechst 33342 from the cells, which leads to intracellular accumulation. All these factors contribute to the enhanced blue fluorescence observed in apoptotic cells.
This kit is suitable for the identification and enumeration of dead cells, such as apoptotic or necrotic cells, by flow cytometry.
Components
A1521084 | Components | 20T | 50T | 100T | Storage | Quantity Per Test | A1521084A | 10× Annexin V Binding Buffer | 5 mL | 10 mL | 20 mL | 2-8℃ | 200 μL/0.5-1.0×10⁵ cells | A11521084B | Annexin V-AF594 | 100 μL | 250 μL | 500 μL | 2-8℃. Store in the dark. | 5 μL/0.5-1.0×10⁵ cells | A1521084C | Hoechst 33342 Staining Solution | 40 μL | 100 μL | 200 μL | 2-8℃. Store in the dark. | 2 μL/0.5-1.0×10⁵ cells |
注: 单次测试推荐染色的细胞数量为 0.5-1.0×10⁵细胞 |
Instruction for use
1. Dilute 10× Binding Buffer to 1× using distilled water (1 mL 10× Binding Buffer + 9 mL ddH2O).
2. Wash cells twice with cold PBS and then resuspend the desired amount of cells in Annexin V Binding Buffer at a concentration of 0.5-1.0×106 cells/mL.
3. Add 5 µl of Annexin-AF594 and 2 µl Hoechst 33342 Staining Solution to 100 µL of the cell suspension
4. Gently vortex the cells and incubate for 10 min at RT (25°C) in the dark.
5. Add 100 µl of 1× Binding Buffer to each assay. Analyze by flow cytometry within 1 hr.
Precautions
1. Please try to avoid light when using to slow down the quenching of fluorescence.
2. Propidium Iodide Solution is toxigenic and mutagenic; handle with care.
3. Due to the calcium dependence of the Annexin V:PS interaction, it is critical to avoid buffers containing EDTA or other calcium chelators during Annexin V experiments.
