Annexin V-PE/7-AAD 凋亡检测试剂盒

产品信息：
应用：流式
储存及稳定性：2℃-8℃避光保存，勿冷冻。
本试剂盒所有组份依保存条件储存，自收到之日起一年有效。
安全注意事项：7-AAD是一种潜在的致癌物。为了您的安全和健康，操作时请穿实验服、戴手套、做好眼睛及面部防
护，避免7-AAD接触皮肤及眼睛。
试剂盒组分：

应用验证：
Annexin V-FITC/7-AAD 凋亡检测试剂盒在喜树碱处理过的Jurkat细胞上做常规验证。Annexin V与磷脂酰丝氨酸（PS）的结合为钙离
子依赖性的，在反应时需使用含钙离子的缓冲液。
Annexin V-FITC/7-AAD 凋亡检测试剂盒未在贴壁细胞上做常规验证。在贴壁细胞的收集过程中，消化细胞容易造成细胞膜损伤，从
而导致产生错误的实验结果。在您的研究中如果用到贴壁细胞，建议提前摸索细胞消化条件，并且尽量避免使用含EDTA的细胞消化
液，以免产生错误的实验结果。

流式细胞术检测Annexin V-FITC染色。Annexin V-FITC及7-AAD在未处理的Jurkat细胞（上图）、4μM喜树碱（Sigma，货号C9911）处
理5小时（中图）及18小时（下图）的Jurkat细胞上的染色结果。未处理的细胞主要为Annexin V-FITC及7-AAD阴性。喜树碱处理5小时后，
主要出现两群细胞：未凋亡的活细胞（ Annexin V-FITC 、7-AAD双阴性），早期凋亡的细胞（ Annexin V-FITC 阳性、7-AAD阴性）。
一小部分Annexin V-FITC、7-AAD双阳性的细胞为凋亡晚期及已经死亡的细胞。经过18小时的处理后，凋亡晚期及已经死亡的细胞
（ Annexin V-FITC、7-AAD双阳性）比例明显增加。

Apoptosis is a process of programmed cell death that occurs in multicellular organisms. It is a programmed cell death mechanism characterized by loss of plasma membrane asymmetry and attachment, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, and global mRNA decay. One of the earliest features is the change of plasma membrane.

Annexin V, also known as Annexin A5 (ANXA5), they are abundant intracellular proteins, and several different annexin gene products are expressed in all mammalian cells examined to date. Annexin V belongs to a family of Ca²⁺ binding proteins that undergo reversible Ca²⁺-dependent binding to phospholipids (PLs). In healthy cells, phosphatidylserine (PS) is predominantly located along the cytosolic side of the plasma membrane, upon initiation of apoptosis, the PS is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment, Annexin V has a high affinity for PS, and binds to cells with exposed PS. Annexin V may be conjugated to fluorochromes including FITC, 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.

7-AAD (7-amino-actinomycin D) has a high DNA binding constant and is efficiently excluded by intact cells, can be used in place of propidium iodide (PI) for the exclusion of nonviable cells. Different from PI, the 7-AAD has minimal spectral overlap with phycoerythrin (PE) and fluorescein isothiocyanate (FITC), and can be used in conjunction with PE and FITC-labelled antibodies in mulitcolor analysis.

In early stage apoptosis, the PS was exposed on the cell surface, but the plasma membrane excludes 7-AAD. These cells will stain with Annexin V but not 7-AAD, thus distinguishing cells in early apoptosis (Annexin V positive, 7-AAD negative). In late stage apoptosis, the cell membrane loses integrity thereby allowing 7-AAD access and bind to the DNA, at the same time allowing Annexin V access binding with the PS in the interior of the cell (Annexin V positive, 7-AAD positive). However this assay can't distinguish the cells that died undergone apoptotic with those that have died as a result of necrosis, in either case, the dead cells will stain with both Annexin V and 7-AAD.