Annexin V PE Apop Dtec Kit 100Tst

10X Annexin V Binding Buffer【子货号：51-66121E，包装：50 ml (1 ea)，，运保温度：4°C】

7-AAD【子货号：51-68981E，包装：2.0 ml (1 ea)，，运保温度：4°C】

51-65875X【子货号：51-65875X，包装：0.5 ml (1 ea)，，运保温度：4°C】

细胞凋亡是一种发生在胚胎发育过程以及在机体组织保持稳态的过程中的正常生理过程。凋亡程序包含某些形态学特征，包括细胞膜不对称性和附着、细胞质和细胞核的缩合以及DNA的内部断裂。细胞膜的损失是最早的特征之一。在凋亡细胞中，膜磷脂酰丝氨酸（PS）从细胞膜的内部转移到外部小叶，从而暴露到细胞外部环境。膜联蛋白V是一个35-36 kDa的钙离子依赖性的磷脂结合的蛋白，对于PS的具有高亲和性，可通过暴露的PS结合到细胞上。膜联蛋白V可偶联荧光染料，包括PE。该结合形式仍保留其对于PS的高亲和性，因而可作为一种敏感的探针，用于对正在处于凋亡过程的细胞进行FACS分析。由于PS的外翻发生在凋亡的早期阶段，故PE膜联蛋白V染色相对于基于细胞核的变化的分析技术（例如DNA片段化分析），可在较早的阶段识别细胞凋亡。

 

用PE膜联蛋白V方法染色时，通常将一种活体染料结合使用，例如7-氨基放线菌素（7-AAD），以允许研究者识别早期的凋亡细胞（7-AAD阴性，PE膜联蛋白V阳性）。活细胞具有完整的细胞膜，7-AAD不能渗透进入细胞，而7-AAD可透过死亡和损伤的细胞的细胞膜。例如，PE膜联蛋白V和7-AAD染色均为阴性，则认为是活细胞；PE膜联蛋白V阳性和7-AAD阴性时，认为细胞处于早期凋亡阶段；PE膜联蛋白V和7-AAD染色均为阳性时，可认为细胞处于晚期凋亡阶段或已经死亡。此方法不能区分经历凋亡过程的死细胞与经历死亡过程的死细胞，因为在这两种情况下，死细胞均可被PE膜联蛋白V和PI染色。然而，检测细胞凋亡时，随着时间的推移，细胞可从PE膜联蛋白V和7-AAD阴性（活细胞状态，或检测不到凋亡）状态，演变为PE膜联蛋白V阳性和7-AAD阴性（早期凋亡，细胞膜仍然完整），最后到PE膜联蛋白V和7-AAD阳性（细胞凋亡终末期和死亡）。细胞经历这三个阶段即表明凋亡。与此相反，单次观察显示细胞均为PE膜联蛋白V和7-AAD阳性时，仅可提供细胞消亡过程的较少的信息。

Apoptosis is a normal physiologic process which occurs during embryonic development as well as in maintenence of tissue homeostasis. The apoptotic program is characterized by certain morphologic features, including loss of plasma membrane asymmetry and attachment, condensation of the cytoplasm and nucleus, and internucleosomal cleavage of DNA. Loss of plasma membrane is one of the earliest features. In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment. Annexin V is a 35-36 kDa Ca2+ dependent phospholipid-binding protein that has a high affinity for PS, and binds to cells with exposed PS. Annexin V may be conjugated to fluorochromes including Phycoerythrin (PE). This format retains its high affinity for PS and thus serves as a sensitive probe for flow cytometric analysis of cells that are undergoing apoptosis. Since externalization of PS occurs in the earlier stages of apoptosis, PE Annexin V staining can identify apoptosis at an earlier stage than assays based on nuclear changes such as DNA fragmentation.

 

PE Annexin V staining precedes the loss of membrane integrity which accompanies the latest stages of cell death resulting from either apoptotic or necrotic processes. Therefore, staining with PE Annexin V is typically used in conjunction with a vital dye such as 7-Amino-Actinomycin (7-AAD) to allow the investigator to identify early apoptotic cells (7-AAD negative, PE Annexin V positive). Viable cells with intact membranes exclude 7-AAD, wheras the membranes of dead and damaged cells are permeable to 7-AAD. For example, cells that are considered viable are PE Annexin V and 7-AAD negative; cells that are in early apoptosis are PE Annexin V positive and 7-AAD negative; and cells that are in late apoptosis or already dead are are both PE Annexin V and 7-AAD positive. This assay does not distinguish between cells that have undergone apoptotic death versus those that have died as a result of a necrotic pathway because in either case, the dead cells will stain with both PE Annexin V and 7-AAD. However, when apoptosis is measured over time, cells can be often tracked from PE Annexin V and 7-AAD negative (viable, or no measurable apoptosis), to PE Annexin V positive and 7-AAD negative (early apoptosis, membrane integrity is present) and finally to PE Annexin V and 7-AAD positive (end stage apoptosis and death). The movement of cells through these three stages suggests apoptosis. In contrast, a single observation indicating that cells are both PE Annexin V and 7-AAD positive, in of itself, reveals less information about the process by which the cells underwent their demise.