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FITC Annexin V Apoptosis Detection Kit with PI
上海研卉生物科技有限公司
100T
细胞凋亡(apoptosis)是多细胞动物通过控制机体有害组织发育或清除异常细胞来达到适应生存环境的一种主动机制,是一种自主的有序的死亡过程。细胞表面受体在受到外部死亡信号的刺激后就可以在启动细胞凋亡,其中一类常见的受体是TNF(Tumor Necrosis Factor)超家族,也被称为死亡受体。其它的一些细胞胞内和胞外分子,如FAS、Caspase也都会参与到控制细胞凋亡的途径中。
很多研究都会涉及到细胞凋亡的检测,其中利用荧光素(FITC、PE等)偶联的Annexin V和磷脂酰丝氨酸(phosphotidylserine,PS)特异性结合,并结合PI或7-AAD等非渗透性的核酸染料来判断细胞早期凋亡的情况是最为常见的一种手段。在正常细胞中,PS只分布在细胞膜脂质双分子层的内侧,而在细胞凋亡早期,细胞膜中的PS由脂膜内侧翻向外侧。而Annexin V是一种分子量为35-36kD的Ca依赖性磷脂结合蛋白,能与细胞凋亡过程中翻转到膜外的PS高亲和力特异性结合。再通过PI或7-AAD等染料来判断细胞膜的完整性, 从而区分Annexin V阳性的细胞是早期凋亡细胞还是坏死/晚期凋亡细胞。
BioLegend为细胞凋亡研究提供了多种研究工具,除了Annexin V偶联物以外,还有抗体、功能性重组蛋白等。更多信息请访问:www.biolegend.com
BioLegend's FITC Annexin V Apoptosis Detection Kit with PI has been specifically designed for the identification of apoptotic and necrotic cells.
Annexin V (or Annexin A5) is a member of the annexin family of intracellular proteins that binds to phosphatidylserine (PS) in a calcium-dependent manner. PS is normally only found on the intracellular leaflet of the plasma membrane in healthy cells, but during early apoptosis, membrane asymmetry is lost and PS translocates to the external leaflet. Fluorochrome-labeled Annexin V can then be used to specifically target and identify apoptotic cells. Annexin V Binding Buffer is recommended for use with Annexin V staining. Annexin V binding alone cannot differentiate between apoptotic and necrotic cells. To help distinguish between the necrotic and apoptotic cells we recommend use of our Propidium Iodide Solution (PI). Early apoptotic cells will exclude PI, while late stage apoptotic cells and necrotic cells will stain positively, due to the passage of these dyes into the nucleus where they bind to DNA.
Propidium iodide is a fluorescent dye that binds to DNA. When excited by 488 nm laser light, it can be detected with in the PE/Texas Red® channel with a bandpass filter 610/10. It is commonly used in evaluation of cell viability or DNA content in cell cycle analysis by flow cytometry.
Staining Procedure:
1. Wash cells twice with cold BioLegend's Cell Staining Buffer, and then resuspend cells in Annexin V Binding Buffer at a concentration of 0.25-1.0 x 107 cells/ml.
2. Transfer 100 µl of cell suspension in a 5 ml test tube.
3. Add 5 µl of FITC Annexin V.
4. Add 10 µl of Propidium Iodide Solution.
5. Gently vortex the cells and incubate for 15 min at room temperature (25°C) in the dark.
6. Add 400 µl of Annexin V Binding Buffer to each tube. Analyze by flow cytometry with proper machine settings.
Materials Provided:
0.5 ml of FITC Annexin V
1 ml of Propidium Iodide Solution
50 ml of Annexin V Binding Buffer
Materials Not Included:
Cell Staining Buffer (Cat. No. 420201)
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