适用仪器

样品实验方案

简要概述

1. 用5×10⁵至1×10⁶细胞/ mL的密度制备含测试化合物的细胞
2. 将0.5 µL细胞溶液中加入1 µL 500X TF2-VAD-FMK
3. 将细胞在37°C，5％CO₂培养箱中孵育1-4小时
4. 沉淀细胞并将细胞重悬于0.5 mL检测缓冲液或生长培养基中
5. 使用带有530/30 nm滤光片（FITC通道）的流式细胞仪分析细胞

实验步骤

1.对于每个样品，在0.5 mL温热培养基或自备缓冲液中以5×10⁵至1×10⁶cell/ mL的密度制备细胞。注意：应单独评估每种细胞系，以确定诱导凋亡的佳细胞密度。

2.用测试化合物处理细胞一段时间，以诱导细胞凋亡，并建立阳性和阴性对照。

3.向处理过的细胞中加入1µL 500X TF2-VAD-FMK（组分A）。

4.将细胞在37°C，5％CO₂培养箱中孵育1-4小时。注意：对于贴壁细胞，用0.5 mM EDTA轻轻提起细胞以保持细胞完整，并在与TF2-VAD-FMK孵育之前用含血清的培养基洗涤细胞一次。合适的孵育时间取决于所用的单个细胞类型和细胞浓度。优化每个实验的孵育时间。

5.洗涤并旋转细胞两次。将细胞重悬于0.5 mL分析缓冲液（组分B）或生长培养基中。注意：TF2-VAD-FMK是荧光的；因此，重要的是洗净任何未结合的试剂以除去背景。

6.可选：可以用DNA染色剂标记细胞（例如碘化丙啶或死细胞的7-AAD）。

7.可选：修复细胞。

8.使用带有530/30 nm滤光片的流式细胞仪（FITC通道）检测荧光强度。

相关产品

Angiopoietins Modulate Survival, Migration, and the Components of the Ang-Tie2 Pathway of Chronic Lymphocytic Leukaemia (CLL) Cells In Vitro
Authors: Palma, Luis Mario Aguirre and Flamme, Hanna and Gerke, Iris and Kreuzer, Karl-Anton
Journal: Cancer Microenvironment (2016): 13–26

Hypoxia regulates TRAIL sensitivity of colorectal cancer cells through mitochondrial autophagy.
Authors: Knoll, Gertrud and Bittner, Sebastian and Kurz, Maria and Jantsch, Jonathan and Ehrenschwender, Martin
Journal: Oncotarget (2016)

Promotion of osteointegration under diabetic conditions by tantalum coating-based surface modification on 3-dimensional printed porous titanium implants
Authors: Wang, Lin and Hu, Xiaofan and Ma, Xiangyu and Ma, Zhensheng and Zhang, Yang and Lu, Yizhao and Li, Xiang and Lei, Wei and Feng, Yafei
Journal: Colloids and Surfaces B: Biointerfaces (2016): 440–452

Role of delta-like ligand-4 in chemoresistance against docetaxel in MCF-7 cells
Authors: Wang, Q and Shi, Y and Butler, HJ and Xue, J and Wang, G and Duan, P and Zheng, H
Journal: Human & Experimental Toxicology (2016): 0960327116650006

microRNA-186 inhibits cell proliferation and induces apoptosis in human esophageal squamous cell carcinoma by targeting SKP2
Authors: He, Wei and Feng, Jianfang and Zhang, Yan and Wang, Yuanyuan and Zang, Wenqiao and Zhao, Guoqiang
Journal: Laboratory Investigation (2016): 317–324

Glucose promotes cell proliferation, glucose uptake and invasion in endometrial cancer cells via AMPK/mTOR/S6 and MAPK signaling
Authors: Han, Jianjun and Zhang, Lu and Guo, Hui and Wysham, Weiya Z and Roque, Dario R and Willson, Adam K and Sheng, Xiugui and Zhou, Chunxiao and Bae-Jump, Victoria L
Journal: Gynecologic oncology (2015): 668–675

IL-7 abrogates the immunosuppressive function of human double-negative T cells by activating Akt/mTOR signaling
Authors: Allgäuer, Andrea and Schreiner, Elisabeth and Ferrazzi, Fulvia and Ekici, Arif B and Gerbitz, Armin and Mackensen, Andreas and Völkl, Simon
Journal: The Journal of Immunology (2015): 3139–3148

Insulin improves osteogenesis of titanium implants under diabetic conditions by inhibiting reactive oxygen species overproduction via the PI3K-Akt pathway
Authors: Wang, Lin and Zhao, Xiong and Wei, Bo-yuan and Liu, Yi and Ma, Xiang-yu and Wang, Jian and Cao, Peng-chong and Zhang, Yang and Yan, Ya-bo and Lei, Wei and others
Journal: Biochimie (2015): 85–93

LGL1 modulates proliferation, apoptosis, and migration of human fetal lung fibroblasts
Authors: Zhang, Hui and Sweezey, Neil B and Kaplan, Feige
Journal: American Journal of Physiology-Lung Cellular and Molecular Physiology (2015): L391–L402

t-BHQ Provides Protection against Lead Neurotoxicity via Nrf2/HO-1 Pathway
Authors: Ye, Fang and Li, Xiaoyi and Li, Lili and Yuan, Jing and Chen, Jun
Journal: Oxidative medicine and cellular longevity (2015)

适用仪器

样品实验方案

简要概述

1. 用5×10⁵至1×10⁶细胞/ mL的密度制备含测试化合物的细胞
2. 将0.5 µL细胞溶液中加入1 µL 500X TF5-VAD-FMK
3. 将细胞在37°C，5％CO₂培养箱中孵育1-4小时
4. 沉淀细胞并将细胞重悬于0.5 mL检测缓冲液或生长培养基中
5. 使用带有660/20 nm滤光片的流式细胞仪分析细胞（APC通道）

实验步骤

1.对于每个样品，在0.5mL温热培养基或自备缓冲液中以5×10⁵至1×10⁶cell/ mL的密度制备细胞。注意：应单独评估每种细胞系，以确定诱导凋亡的细胞密度。

2.用测试化合物处理细胞一段时间，以诱导细胞凋亡，并建立阳性和阴性对照。

3.向处理过的细胞中加入1µL 500X TF5-VAD-FMK（组分A）。

4.将细胞在37°C，5％CO₂培养箱中孵育1-4小时。注意：对于贴壁细胞，用0.5 mM EDTA轻轻提起细胞以保持细胞完整，并在与TF5-VAD-FMK孵育之前用含血清的培养基洗涤细胞一次。合适的孵育时间取决于所用的单个细胞类型和细胞浓度。优化每个实验的孵育时间。

5.洗涤并旋转细胞两次。将细胞重悬于0.5 mL分析缓冲液（组分B）或生长培养基中。注意：TF5-VAD-FMK是荧光的；因此，重要的是洗净任何未结合的试剂以除去背景。

6.可选：用DNA染色剂标记细胞（例如绿色DCS标记死细胞，可以用530/30 nm滤光片（FITC通道）检测到。

7.可选：修复细胞。

8.使用带有660/20 nm滤光片（APC通道）的流式细胞仪检测荧光强度。

参考文献

Angiopoietins Modulate Survival, Migration, and the Components of the Ang-Tie2 Pathway of Chronic Lymphocytic Leukaemia (CLL) Cells In Vitro
Authors: Palma, Luis Mario Aguirre and Flamme, Hanna and Gerke, Iris and Kreuzer, Karl-Anton
Journal: Cancer Microenvironment (2016): 13–26

Hypoxia regulates TRAIL sensitivity of colorectal cancer cells through mitochondrial autophagy.
Authors: Knoll, Gertrud and Bittner, Sebastian and Kurz, Maria and Jantsch, Jonathan and Ehrenschwender, Martin
Journal: Oncotarget (2016)

Promotion of osteointegration under diabetic conditions by tantalum coating-based surface modification on 3-dimensional printed porous titanium implants
Authors: Wang, Lin and Hu, Xiaofan and Ma, Xiangyu and Ma, Zhensheng and Zhang, Yang and Lu, Yizhao and Li, Xiang and Lei, Wei and Feng, Yafei
Journal: Colloids and Surfaces B: Biointerfaces (2016): 440–452

Role of delta-like ligand-4 in chemoresistance against docetaxel in MCF-7 cells
Authors: Wang, Q and Shi, Y and Butler, HJ and Xue, J and Wang, G and Duan, P and Zheng, H
Journal: Human & Experimental Toxicology (2016): 0960327116650006

microRNA-186 inhibits cell proliferation and induces apoptosis in human esophageal squamous cell carcinoma by targeting SKP2
Authors: He, Wei and Feng, Jianfang and Zhang, Yan and Wang, Yuanyuan and Zang, Wenqiao and Zhao, Guoqiang
Journal: Laboratory Investigation (2016): 317–324

Glucose promotes cell proliferation, glucose uptake and invasion in endometrial cancer cells via AMPK/mTOR/S6 and MAPK signaling
Authors: Han, Jianjun and Zhang, Lu and Guo, Hui and Wysham, Weiya Z and Roque, Dario R and Willson, Adam K and Sheng, Xiugui and Zhou, Chunxiao and Bae-Jump, Victoria L
Journal: Gynecologic oncology (2015): 668–675

IL-7 abrogates the immunosuppressive function of human double-negative T cells by activating Akt/mTOR signaling
Authors: Allgäuer, Andrea and Schreiner, Elisabeth and Ferrazzi, Fulvia and Ekici, Arif B and Gerbitz, Armin and Mackensen, Andreas and Völkl, Simon
Journal: The Journal of Immunology (2015): 3139–3148

Insulin improves osteogenesis of titanium implants under diabetic conditions by inhibiting reactive oxygen species overproduction via the PI3K-Akt pathway
Authors: Wang, Lin and Zhao, Xiong and Wei, Bo-yuan and Liu, Yi and Ma, Xiang-yu and Wang, Jian and Cao, Peng-chong and Zhang, Yang and Yan, Ya-bo and Lei, Wei and others
Journal: Biochimie (2015): 85–93

LGL1 modulates proliferation, apoptosis, and migration of human fetal lung fibroblasts
Authors: Zhang, Hui and Sweezey, Neil B and Kaplan, Feige
Journal: American Journal of Physiology-Lung Cellular and Molecular Physiology (2015): L391–L402

t-BHQ Provides Protection against Lead Neurotoxicity via Nrf2/HO-1 Pathway
Authors: Ye, Fang and Li, Xiaoyi and Li, Lili and Yuan, Jing and Chen, Jun
Journal: Oxidative medicine and cellular longevity (2015)

适用仪器

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. 添加膜联蛋白V-iFluor 555测定溶液
3. 在室温下孵育30至60分钟
4. 使用带有575/26 nm滤光片（PE通道）的流式细胞仪或带有Cy3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-iFluor 555准备和孵育细胞：

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-iFluor 555（组分A）。

1.5避光保存，于室温下孵育30至60分钟。

1.6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。

1.7使用带有575/26 nm滤光片（PE通道）的流式细胞仪或带有Cy3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575/26 nm滤光片的流式细胞仪（PE通道）定量Annexin V- iFluor 555结合物。

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盖玻片的载玻片上。注意：对于贴壁细胞，建议直接在盖玻片上生长细胞。与Annexin V-iFluor 555孵育后，用测定缓冲液冲洗1-2次，然后将测定缓冲液加到盖玻片上。将玻片上的盖玻片倒置并可视化细胞。与膜联蛋白V-iFluor 555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在荧光显微镜下使用Cy3滤光片组，用Annexin V-iFluor 555分析凋亡细胞。将Nuclear Red DCS1（#17552）添加到细胞后，使用Cy5通道测量细胞活力。质膜上的橙色染色表明膜联蛋白V-iFluor 555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. 添加膜联蛋白V-iFluor 647测定溶液
3. 在室温下孵育30-60分钟
4. 使用带有660/20 nm滤光片（APC通道）的流式细胞仪或带有Cy5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-iFluor 647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-iFluor 647（组分A）。

1.5避光保存，于室温下孵育30至60分钟。

1.6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。

1.7使用带有660/20 nm滤光片的流式细胞仪（APC通道）或带有Cy5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660/20 nm滤光片（APC通道）的流式细胞仪定量膜联蛋白V-iFluor 647的结合物。

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，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盖玻片的载玻片上。 注意：对于贴壁细胞，建议直接在盖玻片上生长细胞。 与Annexin V-iFluor 647一起孵育后，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-iFluor 647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Cy5滤光片组，在荧光显微镜下用Annexin V-iFluor 647分析凋亡细胞。当碘化丙锭添加到细胞中时，通过使用TRITC通道测量细胞活力。质膜上的橙色染色表明膜联蛋白V-iFluor 647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

样品实验方案

简要概述

1. 用5×10⁵至1×10⁶cell/ mL的密度制备含测试化合物的细胞
2. 用70％乙醇固定细胞
3. 将0.5 µL 100X Nuclear Red CCS1和5 µL RNase A加入0.5 mL细胞溶液中
4. 在室温下孵育30-60分钟
5. 使用带有FL2通道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，以诱导凋亡或其他细胞周期功能。

2.对于每个样品，用0.5 mL PBS制备细胞，密度为5×10⁵至1×10⁶细胞/ mL。

2.1对于贴壁细胞：将细胞用胰蛋白酶消化，悬浮在10％FBS培养基中，离心（1000 rpm，5分钟），然后将沉淀重悬于PBS中。

2.2对于悬浮细胞：将细胞离心（1000 rpm，5分钟），并将沉淀物悬浮在PBS（1 mL）中。 注意：应单独评估每种细胞系，以确定诱导凋亡的细胞密度。

3.用70％乙醇固定细胞：

3.1将0.5 mL细胞悬液吸移至1.2 mL无水乙醇中（浓度约为70％）。

3.2在冰上孵育细胞至少2小时（或在-20°C过夜）。在染色之前，细胞可以在-20°C下保存2年。注意：在用单克隆抗体染色细胞表面抗原后，通常将乙醇用于固定，而在用单克隆抗体染色细胞内抗原后，通常将甲醇用于固定。在此过程中，将固定并分析整个细胞。由于仍然存在整个细胞团，因此通常包括使用RNase来任何双链RNA。尽管已经对整个细胞进行了分析，但尝试检测与DNA结合的某些细胞内抗原的尝试可能会失败，因为蛋白质会从透化的细胞中漏出（例如绿色荧光蛋白）。在这些情况下，在酒精固定之前，用PBS中的1％低聚甲醛进行短暂的预固定（在4-6°C下10分钟），将有助于将蛋白质保留在细胞中。

4.用Nuclear Red CCS1染色细胞：

4.1以1000 rpm的速度沉淀细胞5分钟，并用冷PBS至少洗涤一次。

4.2将沉淀物悬浮在0.5 mL测定缓冲液（组分C）中。

4.3加入5 µL 100X Nuclear Red CCS1（组分A）和5 µL 100X RNase A（组分B）。

4.4在室温下孵育细胞30至60分钟。 注意：适当的孵育时间取决于所用的单个细胞类型和细胞浓度。 优化每个实验的孵育时间。

4.5可选：将细胞以1000 rpm离心5分钟，然后将细胞重悬于0.5 mL检测缓冲液（组分B）或自备缓冲液中。

4.6使用FL2通道（Ex / Em = 490/620 nm），通过流式细胞仪检测荧光强度。 

参考文献 

Cell cycle synchronization of Escherichia coli using the stringent response, with fluorescence labeling assays for DNA content and replication
Authors: Ferullo DJ, Cooper DL, Moore HR, Lovett ST.
Journal: Methods (2009): 8

DNA replication, cell cycle progression and the targeted gene repair reaction
Authors: Engstrom JU, Kmiec EB.
Journal: Cell Cycle (2008): 1402

Morin inhibits the growth of human leukemia HL-60 cells via cell cycle arrest and induction of apoptosis through mitochondria dependent pathway
Authors: Kuo HM, Chang LS, Lin YL, Lu HF, Yang JS, Lee JH, Chung JG.
Journal: Anticancer Res (2007): 395

Direct control of cell cycle gene expression by proto-oncogene product ACTR, and its autoregulation underlies its transforming activity
Authors: Louie MC, Revenko AS, Zou JX, Yao J, Chen HW.
Journal: Mol Cell Biol (2006): 3810

Cell cycle markers for live cell analyses
Authors: Easwaran HP, Leonhardt H, Cardoso MC.
Journal: Cell Cycle (2005): 453

Dynamic relocalization of hOGG1 during the cell cycle is disrupted in cells harbouring the hOGG1-Cys326 polymorphic variant
Authors: Luna L, Rolseth V, Hildrestr and GA, Otterlei M, Dantzer F, Bjoras M, Seeberg E.
Journal: Nucleic Acids Res (2005): 1813

Dynamics of relative chromosome position during the cell cycle
Authors: Essers J, van Cappellen WA, Theil AF, van Drunen E, Jaspers NG, Hoeijmakers JH, Wyman C, Vermeulen W, Kanaar R.
Journal: Mol Biol Cell (2005): 769

Cell cycle regulation of the murine 8-oxoguanine DNA glycosylase (mOGG1): mOGG1 associates with microtubules during interphase and mitosis
Authors: Conlon KA, Zharkov DO, Berrios M.
Journal: DNA Repair (Amst) (2004): 1601

Description of a flow cytometry approach based on SYBR-14 staining for the assessment of DNA content, cell cycle analysis, and sorting of living normal and neoplastic cells
Authors: Nunez R, Garay N, Villafane C, Bruno A, Lindgren V.
Journal: Exp Mol Pathol (2004): 29

Differential roles of STAT1alpha and STAT1beta in fludarabine-induced cell cycle arrest and apoptosis in human B cells
Authors: Baran-Marszak F, Feuillard J, Najjar I, Le Clorennec C, Bechet JM, Dusanter-Fourt I, Bornkamm GW, Raphael M, Fagard R.
Journal: Blood (2004): 2475