背景介绍
膜联蛋白（Annexins）是一类钙离子依赖型磷脂结合蛋白，能特异性结合磷脂酰丝氨酸（PS）。在正常生理状态下，PS只分布在细胞膜脂质双层的内侧。细胞凋亡早期， PS由脂膜内侧翻向外侧，在磷脂双分子层中的不对称分布丧失，从而使细胞成为被吞噬的目标。一旦PS暴露在膜的外表面，它便可在钙离子存在的情况下，被荧光标记的膜联蛋白V（Annexin V）检测到。
荧光染料Hoechst33342能少许进入正常细胞膜，使其染上低蓝色，而凋亡细胞的膜通透性增强，因此进入凋亡细胞中的Hoechst33342比正常细胞的多，荧光强度要比正常细胞中要高，此外，凋亡细胞的染色体DNA的结构发生了改变从而使该染料能更有效地与DNA结合，并且凋亡细胞膜上的p-糖蛋白泵功能受到损伤不能有效地将Hoechst33342排出到细胞外使之在细胞内积累增加等都使凋亡细胞的蓝色荧光增强。
本试剂盒适用于通过流式细胞术对死亡细胞（如凋亡或坏死细胞）进行鉴定和计数。
产品组分
A1521084 | 组份 | 20T | 50T | 100T | 储存条件 | 单次测试使用量 | A1521084A | 10× Annexin V结合缓冲液 | 5 mL | 10 mL | 20 mL | 2-8℃ | 200 μL/0.5-1.0×10⁵细胞 | A11521084B | Annexin V-AF594 | 100 μL | 250 μL | 500 μL | 2-8℃, 避光. | 5 μL/0.5-1.0×10⁵细胞 | A1521084C | Hoechst 33342染色液 | 40 μL | 100 μL | 200 μL | 2-8℃, 避光. | 2 μL/0.5-1.0×10⁵细胞 |
注: 单次测试推荐染色的细胞数量为 0.5-1.0×10⁵细胞 |
使用说明
1. 使用前需将10× 结合缓冲液用去离子水稀释至1×（1 mL 10×缓冲液加9 mL去离子水）；
2. 预冷PBS清洗细胞两次后，按照0.5-1.0×106/mL的密度用1× Annexin V结合缓冲液重悬；
3. 每100 μL细胞悬液中加入5 μL Annexin V-AF594及2 μL Hoechst 33342染色液；
4. 轻柔涡旋混匀细胞，室温避光孵育10 min；
5. 加入100 μL结合缓冲液，1h内完成上机分析。
注意事项
1. 使用时请避光以减缓荧光淬灭；
2. PI染料具有具有剧毒性和致突变性，操作时需谨慎；
3. 由于膜联蛋白V（Annexin V）与磷脂酰丝氨酸（PS）的结合具有钙依赖性，因此在Annexin V实验中务必避免使用含EDTA或其他钙螯合剂的缓冲液。
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.
ZJ26F0838251
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