Annexin V-PE/7-AAD细胞凋亡检测试剂盒是用红色荧光染料PE（Phycoerythrin）标记的Annexin V作为探针，来检测细胞早期凋亡的发生，可用荧光显微镜、流式细胞仪或其他荧光检测设备进行检测。
其检测原理为：在正常的活细胞中，磷脂酰丝氨酸（phosphotidylserine，PS）位于细胞膜的内侧，但在早期凋亡的细胞中，PS 从细胞膜的内侧翻转到细胞膜的表面，暴露在细胞外环境中。Annexin-Ⅴ（膜联蛋白-V）是一种分子量为35-36KD的Ca2+ 依赖性磷脂结合蛋白，能与PS高亲和力结合。可通过细胞外侧暴露的磷脂酰丝氨酸与凋亡早期细胞的胞膜结合。
另外，本试剂盒中提供的7-AAD可用来区分存活的早期细胞和坏死或晚期凋亡细胞。7-AAD是一种核酸染料，同PI有着相似的荧光特性，但其发射光谱较PI窄，对其他检测通道的干扰更小，在多色荧光分析中是PI的最佳替代品，可与Annexin V联合使用。该染料不能透过正常细胞或早期凋亡细胞的完整的细胞膜，但可穿透晚期凋亡细胞或者坏死细胞并与其内的DNA结合。因此将Annexin V-PE与7-AAD联合使用时，7-AAD则被排除在活细胞（Annexin V-/7-AAD-）和早期凋亡细胞（Annexin V+/7-AAD-）之外，而晚期凋亡细胞和坏死细胞同时被Annexin V-PE和7-AAD结合染色呈现双阳性（Annexin V+/7-AAD+）。
检测便捷、准确
冰袋（wet ice）运输。
本试剂盒中所有组分均在4 ℃保存，勿冰冻。Annexin V-PE、7-AAD需避光保存。一年有效。
Q:这两个试剂用什么通道检测？仪器是贝克曼的。
A: Annexin V-PE 的激发波长Ex=488nm；发射波长Em=578 nm，发出橙红色荧光，建议使用 FL2 通道检测；7-AAD 的激发波长 Ex=546 nm；发射波长 Em=647 nm，发出红色荧光， 建议使用 FL3 通道检测。
Q: Annexin V-PE 染色失败或者阳性率偏低。
A:首先要确定实验中使用的诱导剂是否能产生凋亡。可通过设定确切凋亡诱导效果的阳性药物对照来排除这一情况。Annexin V-PE 染色失败，最常见的实验操作原因是贴壁细胞消化不当。Annexin V 跟 PS 的结合需要 Ca2+，Binding Buffer 中含有 2.5 mM 的 Ca2+，含 EDTA 的胰酶消化会影响染色，建议使用无EDTA 的胰酶。若使用含EDTA 的胰酶，必须通过洗涤步骤彻底去除EDTA。用 PBS 洗涤细胞沉淀后，应尽量去除残余液体。残留PBS 中的磷酸根，会形成磷酸钙沉淀。Binding Buffer 的瓶盖要紧闭，防止空气中的 CO2 进入后形成碳酸钙沉淀，减少游离Ca2+，导致实验失败。接触其他缓冲液如吸取PBS 后不更换枪头也会导致游离的 Ca2+减少。一些细胞其细胞膜上 PS 的密度较低，染色效果很差，此时建议更换细胞株或者采用 TUNEL 试剂盒检测凋亡。如果是贴壁细胞，药物诱导后漂浮的细胞也要收集，这部分细胞往往是凋亡阳性的细胞，丢弃会造成阳性结果偏低。
Q: 假阳性
A: 实验中发现未经诱导凋亡的对照细胞经染色后Annexin V-PE/7-AAD 双阳性比例过高，造成这种结果的原因可能是细胞本身活力低。建议用台盼蓝染色计算细胞活力，阴性对照台盼蓝拒染的细胞应大于 95%。若细胞活力低，建议重新复苏细胞，通常刚复苏的细胞至少应传代 2- 3 次以后才能进行实验。另一可能是细胞操作不当引起，操作过程中吹打细胞过于剧烈，贴壁细胞消化过程中消化时间过长，均可能导致细胞膜破坏，出现假阳性。此外，一般情况下诱导几个小时就可以出现早期凋亡，因此通常不需要处理大于 48 h 以上；诱导时间过长会使营养物质耗尽，导致细胞状态差，假阳性结果偏高。
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