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产品描述
Annexin V-FITC/PI细胞凋亡检测试剂盒是用FITC标记的Annexin V作为探针，来检测细胞早期凋亡的发生。
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35-36 kDa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，本试剂盒中还提供了碘化丙啶（Propidium Iodide，PI）用来区分存活的早期细胞和坏死或晚期凋亡细胞。PI是一种核酸染料，它不能透过正常细胞或早期凋亡细胞的完整的细胞膜，但可以透过凋亡晚期和坏死细胞的细胞膜而使细胞核染红。因此，将Annexin V与PI联合使用时，PI 则被排除在活细胞（Annexin V-/PI-）和早期凋亡细胞（Annexin V+/PI-）之外，而晚期凋亡细胞和坏死细胞同时被FITC 和PI 结合染色呈现双阳性（Annexin V+/PI+）。
本试剂盒可用于流式细胞仪、荧光显微镜进行检测。
产品组分
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编号 |
组分 |
产品编号/规格 |
||
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40302ES20（20T） |
40302ES50（50T） |
40302ES60（100T） |
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40302-A |
Annexin V-FITC |
100 μL |
250 μL |
500 μL |
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40302-B |
PI Staining Solution |
200 μL |
500 μL |
1.0 mL |
|
40302-C |
1×Binding Buffer |
10 mL |
25 mL |
50 mL |
运输与保存方法
冰袋（wet ice）运输。-20℃避光保存，避免反复冻融，一年有效。
【注】：如果需要在短时间内多次重复使用，可以在4℃避光保存，半年有效。
注意事项
1）由于细胞凋亡是一个快速的过程，建议样品在染色后1小时之内进行分析。
2） 对于贴壁细胞，消化是一个关键步骤。贴壁细胞诱导细胞凋亡时如有漂浮细胞，需收集漂浮细胞和贴壁细胞后合并染色。处理贴壁细胞时要小心操作，尽量避免人为的损伤。胰酶消化时间过短，细胞需要用力吹打才能脱落，容易造成细胞膜的损伤；PI摄入过多，消化时间过长，细胞膜同样易造成损伤，甚至会影响细胞膜上磷脂酰丝氨酸与Annexin V-FITC的结合。消化时将胰酶铺满孔板底后，轻摇使胰酶与细胞充分接触，然后倒掉大部分胰酶，利用剩余少量胰酶再消化一段时间，待细胞间空隙增大，瓶底呈花斑状即可终止。在消化液中尽量不用EDTA，EDTA会影响Annexin V与PS的结合。
3）如果样品来源于血液，请务必除去血液中的血小板。因为血小板含有PS，能与Annexin V结合，从而干扰实验结果。可以使用含有EDTA的缓冲剂并在200 g离心洗去血小板。
4）试剂在开盖前请短暂离心，将盖内壁上的液体甩至管底，避免开盖时液体洒落。
5）Annexin V-FITC和PI是光敏物质，在操作时请注意避光。
6）本产品仅作科研用途！
操作方法
1.1 样品染色
1）悬浮细胞：300 g，4℃离心5 min收集细胞。
贴壁细胞：用不含EDTA的胰酶消化后，300 g，4℃离心5 min收集细胞。胰酶消化时间不宜过长，以防引起假阳性。
2）用预冷的PBS洗涤细胞2次，每次均需300 g，4℃离心5 min。收集1～5×105细胞。
3）吸弃PBS，加入100 μL 1×Binding Buffer重悬细胞。
4）加入5 μL Annexin V-FITC和10 μLPI Staining Solution，轻轻混匀。
5）避光、室温反应10-15 min。
6）加入400 μL 1×Binding Buffer，混匀后放置于冰上，样品在1小时内用流式细胞仪或荧光显微镜检测。
【注】：为了避免洗涤细胞时损失细胞，在吸液时可以用大的Tip头套上小的Tip头吸液。
1.2 样品分析
A．流式细胞仪分析：
FITC最大激发波长为488 nm，最大发射波长525 nm，FITC的绿色荧光在FL1通道检测；PI-DNA复合物的最大激发波长为535 nm，最大发射波长为615 nm，PI的红色荧光在FL2或FL3通道检测。用CellQuest等软件进行分析，绘制双色散点图（two-color dot plot），FITC为横坐标，PI为纵坐标。典型的实验中，细胞可以分成三个亚群，活细胞仅有很低强度的背景荧光，早期凋亡细胞仅有较强的绿色荧光，晚期凋亡细胞有绿色和红色荧光双重染色。
B．荧光显微镜分析：
1）滴一滴用Annexin V-FITC/PI双染的细胞悬液于载玻片上，并用盖玻片盖上细胞。
【注】：对于贴壁细胞，可直接用盖玻片培养细胞并诱导细胞凋亡。
2）在荧光显微镜下用双色滤光片观察。Annexin V-FITC荧光信号呈绿色，PI荧光信号呈红色。
相关产品
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产品名称 |
货号 |
规格 |
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40301ES50 |
50 T |
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40301ES60 |
100 T |
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40303ES20 |
20 T |
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40303ES50 |
50 T |
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40303ES60 |
100 T |
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40304ES20 |
20 T |
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40304ES50 |
50 T |
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40304ES60 |
100 T |
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40305ES20 |
20 T |
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40305ES50 |
50 T |
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40305ES60 |
100 T |
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40310ES20 |
20 T |
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40310ES50 |
50 T |
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40310ES60 |
100 T |
HB220609
Q：Annexin V 和 JC-1、Tunel 细胞凋亡检测的区别？
A: Annexin V 是检测细胞早期凋亡的试剂，JC-1 是检测细胞中期凋亡的试剂、Tunel 是检测细胞晚期凋亡的试剂。
Q：Annexin V 和JC-1、Tunel 细胞凋亡检测的可以应用到植物或是细菌（原核生物） 吗？
A：可以，但是需要制备原生质体，因为植物细胞或是细菌（原核生物）含有细胞壁，具体的染液使用剂量只需浸没细胞即可，染色时间对于不同细胞有一定的不同。
Q：40302ES Annexin V-FITC/PI 细胞凋亡检测试剂盒里的PI的浓度是多少呢？
A：20ug/ml。
Q:实验结果如何判断？
A：活细胞（Annexin V-/PI-）
早期凋亡细胞（Annexin V+/PI-）
晚期凋亡细胞和坏死细胞呈现双阳性（Annexin V+/PI+）
裸核（Annexin V-/PI+）
Q: Annexin V和TUNEL有什么区别？
A：末端脱氧核苷酸转移酶 dUTP 缺口末端标记 (TUNEL) 是一种染色方法，用于识别细胞内 DNA 片段化位点——晚期细胞凋亡的标志性特征。 它使用酶末端脱氧核苷酸转移酶 (TdT) 将修饰的 dNTP（例如 dUTP）连接到片段化 DNA 链的 3'-羟基末端。 dNTPs 通常用荧光团修饰以促进量化和/或可视化。
Annexin V 染色通过结合由于细胞膜不对称性丧失而暴露在细胞外的 PS 残基来识别细胞凋亡的早期阶段。 Annexin V 通常用 FITC 等荧光团标记，以促进凋亡细胞的检测。
Q: 1×Binding Buffer 的成分是什么？可以用什么替代？
A：成分信息保密；可以用PBS或者Hanks缓冲液代替，但是要补加2.5mM 的CaCl2。
Q: 是否可以仅使用Annexin V-FITC进行检测而不检测PI？
A：可以的。对于特殊的检测样本，如红细胞无细胞核，如使用药物Doxorubicin（阿霉素）对PI检测存在干扰，对于这两种情况可以仅使用Annexin V-FITC进行检测。
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