适用仪器

样品实验方案

简要概述

1. 用测试化合物制备细胞（200 µL /样品）
2. 添加膜联蛋白V-mFluor Violet 550测定溶液
3. 在室温下孵育30至60分钟
4. 使用带有525/50 nm滤光片（Pacific Orange通道）的流式细胞仪或带有紫光滤光片组的荧光显微镜分析细胞

实验步骤

1.用膜联蛋白V-mFluor Violet 550制备和孵育细胞：

1.1用测试化合物处理细胞一段时间（对于用星形孢菌素处理过的Jurkat细胞，需要4-6小时）以诱导细胞凋亡。

1.2离心细胞以获得1-5×10⁵细胞/管。

1.3将细胞重悬于200 µL分析缓冲液（组分B）中。

1.4向细胞中加入2 µL Annexin V-mFluor Violet 550（组分A）。

1.5可选：向坏死细胞中加入2 µL 100X碘化丙啶（组分C）。

1.6避光保存，于室温下孵育30至60分钟。

1.7在使用流式细胞仪或荧光显微镜分析细胞之前，添加300 µL分析缓冲液（组分B）以增加体积。

1.8使用带有525/50 nm滤光片的流式细胞仪（Pacific Orange通道）或带有紫色滤光片组的荧光显微镜检测荧光强度。

2.通过使用流式细胞仪进行分析：

2.1使用带有525/50 nm滤光片（Pacific Orange通道）的流式细胞仪，定量膜联蛋白V-mFluor Violet 550的结合。 将碘化丙啶加入细胞后，使用610/20 nm滤光片（PE-Texas Red 通道）检测细胞活力。 

3.通过使用荧光显微镜进行分析：

3.1孵育后用移液管吸移细胞悬液，用测定缓冲液冲洗1-2次，然后用测定缓冲液重悬细胞。

3.2将细胞添加到载玻片上。注意：对于贴壁细胞，建议直接在盖玻片上生长细胞。与膜联蛋白V-mFluor Violet 550孵育后，用测定缓冲液冲洗1-2次，然后将测定缓冲液加回到盖玻片上。将玻片上的盖玻片倒置并可视化细胞。与膜联蛋白V-mFluor Violet 550孵育后，还可以将细胞固定在2％甲醛中，并在显微镜下观察。

3.3使用Violet滤光片组在荧光显微镜下用膜联蛋白V-mFluor Violet 550分析凋亡细胞。当碘化丙锭添加到细胞中时，使用TRITC通道检测细胞活力。质膜上的橙色染色表明膜联蛋白V-mFluor Violet 550与细胞表面的PS结合。

参考文献

In situ polymerizable hydrogel incorporated with specific pathogen-free porcine platelet-rich plasma for the reconstruction of the corneal endothelium
Authors: Lin, Yung-Kai and Sharma, Ruchi and Ma, Hsu and Chen, Wen-Shyan and Yao, Chao-Ling
Journal: Journal of the Taiwan Institute of Chemical Engineers (2017)

Novel regulations of MEF2-A, MEF2-D, and CACNA1S in the functional incompetence of adipose-derived mesenchymal stem cells by induced indoxyl sulfate in chronic kidney disease
Authors: Do, Duyen Thi and Phan, Nam Nhut and Wang, Chih-Yang and Sun, Zhengda and Lin, Yen-Chang
Journal: Cytotechnology (2016): 2589–2604

Shear stress-induced alteration of epithelial organization in human renal tubular cells
Authors: Maggiorani, Damien and Dissard, Romain and Belloy, Marcy and Saulnier-Blache, Jean-Sébastien and Casemayou, Audrey and Ducasse, Laure and Grès, S and ra and Bellière, Julie and Caubet, Cécile and Basc and s, Jean-Loup and others
Journal: PloS one (2015): e0131416

Targeting a G-protein-coupled receptor overexpressed in endocrine tumors by magnetic nanoparticles to induce cell death
Authors: Sanchez, Claire and El Hajj Diab, Darine and Connord, Vincent and Clerc, Pascal and Meunier, Etienne and Pipy, Bernard and Payré, Bruno and Tan, Reasmey P and Gougeon, Michel and Carrey, Julian and others
Journal: Acs Nano (2014): 1350–1363

Glycogen synthase kinase-3 and Omi/HtrA2 induce annexin A2 cleavage followed by cell cycle inhibition and apoptosis
Authors: Wang CY, Lin YS, Su WC, Chen CL, Lin CF.
Journal: Mol Biol Cell (2009): 4153

Gold fluorescent annexin A5 as a novel apoptosis detection tool
Authors: Kurschus FC, Pal PP, Baumler P, Jenne DE, Wiltschi B, Budisa N.
Journal: Cytometry A (2009): 626

Detection of apoptosis induced by new type gosling viral enteritis virus in vitro through fluorescein annexin V-FITC/PI double labeling
Authors: Chen S, Cheng AC, Wang MS, Peng X.
Journal: World J Gastroenterol (2008): 2174

Evaluation of annexin V and Calcein-AM as markers of mononuclear cell apoptosis during human immunodeficiency virus infection
Authors: Palma PF, Baggio GL, Spada C, Silva RD, Ferreira SI, Treitinger A.
Journal: Braz J Infect Dis (2008): 108

Measurement of annexin V uptake and lactadherin labeling for the quantification of apoptosis in adherent Tca8113 and ACC-2 cells
Authors: Hu T, Shi J, Jiao X, Zhou J, Yin X.
Journal: Braz J Med Biol Res (2008): 750

Rhodamine B isothiocyanate doped silica-coated fluorescent nanoparticles (RBITC-DSFNPs)-based bioprobes conjugated to Annexin V for apoptosis detection and imaging
Authors: Shi H, He X, Wang K, Yuan Y, Deng K, Chen J, Tan W.
Journal: Nanomedicine (2007): 266

适用仪器

适用仪器

样品实验方案

简要概述

1. 用测试化合物制备细胞（200 µL /样品）
2. 添加Apopxin 深红色测定溶液
3. 在室温下孵育30-60分钟
4. 使用具有FL4通道（Ex / Em = 647/660 nm的流式细胞仪）分析细胞

实验步骤

1.用测试化合物处理细胞所需的时间（喜树碱处理过的Jurkat细胞需要4-6小时）以诱导凋亡。 

2.离心细胞以获得1-5×10⁵细胞/管。

3.将细胞重悬于200 µL分析缓冲液（组分B）中。

4.向细胞中加入2 µL Apopxin 深红色溶液（组分A）。

5.避光保存，于室温下孵育30至60分钟。

6.可选：在使用流式细胞仪分析细胞之前，添加200至300 µL分析缓冲液（组分B）以增加体积。

7.使用带有FL4通道的流式细胞仪（Ex / Em = 647/660 nm）检测荧光强度。

参考文献

Apoptosis of human Burkitt’s lymphoma cells induced by 2-N,N-diethylaminocarbonyloxymethyl-1-diphenylmethyl-4-(3,4,5-trimethoxybe nzoyl) piperazine hydrochloride (PMS-1077)
Authors: Wang WD, Xu XM, Chen Y, Jiang P, Dong CZ, Wang Q.
Journal: Arch Pharm Res (2009): 1727

Detection of apoptosis based on the interaction between annexin V and phosphatidylserine
Authors: Liu T, Zhu W, Yang X, Chen L, Yang R, Hua Z, Li G.
Journal: Anal Chem (2009): 2410

Dynamic analysis of apoptosis using cyanine SYTO probes: from classical to microfluidic cytometry
Authors: Wlodkowic D, Skommer J, Faley S, Darzynkiewicz Z, Cooper JM.
Journal: Exp Cell Res (2009): 1706

Eurycomanone induce apoptosis in HepG2 cells via up-regulation of p53
Authors: Zakaria Y, Rahmat A, Pihie AH, Abdullah NR, Houghton PJ.
Journal: Cancer Cell Int (2009): 16

Evaluation of cell surface expression of phosphatidylserine in ovarian carcinoma effusions using the annexin-V/7-AAD assay: clinical relevance and comparison with other apoptosis parameters
Authors: Dong HP, Holth A, Kleinberg L, Ruud MG, Elstr and MB, Trope CG, Davidson B, Risberg B.
Journal: Am J Clin Pathol (2009): 756

Glycogen synthase kinase-3 and Omi/HtrA2 induce annexin A2 cleavage followed by cell cycle inhibition and apoptosis
Authors: Wang CY, Lin YS, Su WC, Chen CL, Lin CF.
Journal: Mol Biol Cell (2009): 4153

Gold fluorescent annexin A5 as a novel apoptosis detection tool
Authors: Kurschus FC, Pal PP, Baumler P, Jenne DE, Wiltschi B, Budisa N.
Journal: Cytometry A (2009): 626

Induction of apoptosis in sonoporation and ultrasonic gene transfer
Authors: Miller DL, Dou C.
Journal: Ultrasound Med Biol (2009): 144

Mobilization of lysosomal calcium regulates the externalization of phosphatidylserine during apoptosis
Authors: Mirnikjoo B, Balasubramanian K, Schroit AJ.
Journal: J Biol Chem (2009): 6918

Peptidic targeting of phosphatidylserine for the MRI detection of apoptosis in atherosclerotic plaques
Authors: Burtea C, Laurent S, Lancelot E, Ballet S, Murariu O, Rousseaux O, Port M, V and er Elst L, Corot C, Muller RN.
Journal: Mol Pharm (2009): 1903