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（100 µL /孔/ 96孔板或25 µL /孔/ 384孔板）
2. 加入等量的Apopxin Orange工作溶液
3. 在室温下孵育1小时
4. 在Ex / Em = 540/590 nm（截止= 570 nm）或使用Cy3滤光片的荧光显微镜下检测荧光强度（底部读取模式）

溶液配制

工作溶液配制

将10μL的100X Apopxin 橙色（组分A）添加到1 mL的测定缓冲液（组分B）中，并充分混合以制成Apopxin Orange工作溶液。 注意： 100μLApopxin Orange工作溶液足以用于一个孔。现用现配避免长时间放置。

操作步骤

1.通过向PBS或所需的缓冲液中加入10ul /孔（96孔板）或2.5 µL /孔（384孔板）的10X测试化合物储备溶液来处理细胞。 对于空白孔（不含细胞的培养基），添加相同量的化合物缓冲液。

2.将细胞板在5％CO₂、37°C的培养箱中孵育（对于喜树碱处理过的Jurkat细胞，需要4-6小时）以诱导凋亡。

3.在每个孔中添加100 µL /孔（96孔板）或25 µL /孔（384孔板）的Apopxin Orange工作溶液。

4.在避光条件下，于室温下孵育细胞板至少1小时。

5.以800 rpm的速度离心细胞板（特别是非贴壁细胞）2分钟（制动）。

6.使用荧光酶标仪（Ext / Em = 540/590 nm（截止= 570 nm））或使用带有Cy3®滤光片的荧光显微镜对图像进行荧光强度检测。

图示

图1.用Cell Meter 磷脂酰丝氨酸细胞凋亡检测试剂盒染色的Costar黑色96孔板中Jurkat细胞的荧光图像。 不使用（左）/使用20 µM喜树碱（右）处理Jurkat细胞5小时。 使用具有通道的荧光显微镜测量荧光强度。

参考文献

Physiological effects of the herbicide glyphosate on the cyanobacterium Microcystis aeruginosa
Authors: Wu, Liang and Qiu, Zhihao and Zhou, Ya and Du, Yuping and Liu, Chaonan and Ye, Jing and Hu, Xiaojun
Journal: Aquatic Toxicology (2016): 72–79

Tumor-selective mitochondrial network collapse induced by atmospheric gas plasma-activated medium.
Authors: Saito, Kosuke and Asai, Tomohiko and Fujiwara, Kyoko and Sahara, Junki and Koguchi, Haruhisa and Fukuda, Noboru and Suzuki-Karasaki, Miki and Soma, Masayoshi and Suzuki-Karasaki, Yoshihiro
Journal: Oncotarget (2016)

Inhibition of malignant phenotypes of human osteosarcoma cells by a gene silencer, a pyrrole–imidazole polyamide, which targets an E-box motif
Authors: Taniguchi, Masashi and Fujiwara, Kyoko and Nakai, Yuji and Ozaki, Toshinori and Koshikawa, Nobuko and Toshio, Kojima and Kataba, Motoaki and Oguni, Asako and Matsuda, Hiroyuki and Yoshida, Yukihiro and others
Journal: FEBS open bio (2014): 328–334

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

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

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

Suicidal membrane repair regulates phosphatidylserine externalization during apoptosis
Authors: Mirnikjoo B, Balasubramanian K, Schroit AJ.
Journal: J Biol Chem (2009): 22512

Trivalent methylated arsenical-induced phosphatidylserine exposure and apoptosis in platelets may lead to increased thrombus formation
Authors: Bae ON, Lim KM, Noh JY, Chung SM, Kim SH, Chung JH.
Journal: Toxicol Appl Pharmacol (2009): 144

Discovery of a phosphatidylserine-recognizing peptide and its utility in molecular imaging of tumour apoptosis
Authors: Thapa N, Kim S, So IS, Lee BH, Kwon IC, Choi K, Kim IS.
Journal: J Cell Mol Med (2008): 1649