膜联蛋白V-FITC检测试剂盒

膜联蛋白V试剂盒可通过荧光检测与凋亡细胞结合的膜联蛋白V。细胞凋亡，或称程序性细胞死亡，是一个重要的过程，生物体通过该过程确保移除严重受损或突变的细胞。此过程受到蛋白质复杂相互作用的严格调控。

在细胞凋亡的早期阶段，细胞质膜发生重排，导致细胞膜磷脂不对称性改变。位于细胞膜内的磷脂酰丝氨酸（PS）会发生易位并展示在质膜外侧（Fadeel and Xue, 2009）。膜联蛋白V是由ANXA5基因编码的一种蛋白质，在Ca²⁺存在下对PS具有高结合亲和力，因此被用作检测早期凋亡细胞的工具。

制备本产品所使用的偶联方案未改变膜联蛋白V固有的磷脂结合特性。该方案旨在简便、快速地测量悬浮细胞样本中的细胞凋亡。

储存信息

在+4°C下储存。请勿冷冻。
本产品应未经稀释储存。

 

应用

据报道，本产品适用于以下应用。此信息源自我们实验室的内部测试、同行评审的出版物或原研者的个人交流。请参阅所示参考文献以获取更多信息。有关通用实验方案建议，请访问抗体实验方案页面。

应用名称	已验证	最小稀释度	最大稀释度
流式细胞术

若本产品未在特定技术中进行测试，这并不一定排除其在该程序中的使用。建议的工作稀释度仅供参考。建议用户在其自身系统中使用适当的阴性/阳性对照对产品进行滴定。

使用说明

膜联蛋白V-FITC

1) 用蒸馏水将10x结合缓冲液稀释至1x（1 ml 10x结合缓冲液 + 9 ml 蒸馏水）。

2) 离心细胞并用PBS重悬，将细胞密度调整至1 x 10⁶ 个细胞/ml。

3) 将100 μl细胞转移至96孔平底板。

4) 离心细胞并用95 μl 1x结合缓冲液重悬。

5) 加入5 μl FITC标记的膜联蛋白V。

6) 混匀，在室温避光条件下孵育10分钟。

7) 离心细胞并用200 μl 1x结合缓冲液重悬。

8) 用200 μl 1x结合缓冲液重悬细胞，并加入预先优化好浓度的活力染料（例如，1 μg/ml的DAPI或PI）。

9) 立即进行流式细胞术分析。若无法立即分析，在实验设计允许的情况下，可在2-8°C避光条件下储存最多4小时。

 

FAQ

1. 该试剂盒适用于检测哪种类型的细胞凋亡？
该试剂盒适用于检测早期凋亡细胞，原理是检测凋亡早期外翻至细胞膜外的磷脂酰丝氨酸。

2. 该试剂盒推荐用于哪种实验技术？
根据产品信息，该试剂盒已报道可用于流式细胞术分析。

3. 试剂应如何储存？
产品应在+4°C下储存，请勿冷冻，且应未经稀释保存。

4. 染色后的样本可以保存多久？
染色后若无法立即分析，在实验设计允许的情况下，可在2-8°C避光条件下保存最多4小时。

5. 实验操作中需要注意什么？
孵育步骤需要在室温避光条件下进行。建议用户根据自身实验体系，使用适当的阴/阳性对照对试剂进行滴定以确定最佳使用条件。

Annexin V Kit allows fluorescent detection of annexin V bound to cells undergoing apoptosis. Apoptosis, or programmed cell death, is an essential process, by which organisms ensure that critically damaged or mutated cells are removed. This process is highly regulated by a complex interplay of proteins.

During the early stages of apoptosis, the plasma membrane of a cell is rearranged, resulting in changes to the cell membrane’s phospholipid asymmetry. Phosphatidylserine (PS), a phospholipid located within the cell membrane, is translocated and displayed on the outer plasma membrane (Fadeel and Xue, 2009). Annexin V; a protein encoded by the ANXA5 gene, has a high binding affinity for PS in the presence of Ca²⁺, and is therefore used as a tool for detecting early apoptotic cells.

The conjugation protocol used to prepare this product has not changed the native phospholipid binding properties of annexin V. This protocol is designed to measure apoptosis easily and quickly in a sample of suspended cells.

Storage Information

Store at +4°C. DO NOT FREEZE.
This product should be stored undiluted.

 

Applications

This product has been reported to work in the following applications. This information is derived from testing within our laboratories, peer-reviewed publications or personal communications from the originators. Please refer to references indicated for further information. For general protocol recommendations, please visit the antibody protocols page.

Application Name	Verified	Min Dilution	Max Dilution
Flow Cytometry

Where this product has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. Suggested working dilutions are given as a guide only. It is recommended that the user titrates the product for use in their own system using appropriate negative/positive controls.

Instructions For Use

ANNEXINF

1) Dilute the 10x Binding Buffer to 1x in distilled water (1 ml 10x Binding Buffer + 9 ml distilled water).

2) Centrifuge and resuspend cells in PBS, adjusting to a cell density of 1 x 10⁶ cells/ml.

3) Transfer 100 μl cells to a 96-well flat bottom plate.

4) Centrifuge and resuspend cells in 95 μl 1x Binding Buffer.

5) Add 5 μl of FITC conjugated Annexin V

6) Mix and incubate for 10 minutes at room temperature in the dark.

7) Centrifuge and resuspend cells in 200 μl of 1x Binding Buffer.

8) Resuspend cells in 200 μl of 1x Binding Buffer and add viability dye of choice at previously optimized concentration (e.g. DAPI or PI at 1 μg/ml).

9) Analyze by flow cytometry immediately. If not analyzed immediately, store at 2-8°C in the dark for maximum 4 hours, experimental design permitting.