Name: colorectal cancer cell surface protein profiling using an antibody microarray and fluorescence multiplexing
Uploaded: 2011-09-25T14:00:00
Description: Watch this Scientific Journal Video about Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing at JoVE.com

我们感谢的皇家阿尔弗雷德王子“和”儿童权利公约“和正常的肠道黏膜的新鲜样品收集的协和遣返医院解剖病理学实验室的工作人员。这项工作是由癌症研究所的新南威尔士转化项目资助资助。

<img src="/files/ftp_upload/3322/3322fig1.jpg" alt="图1" /> 图1。暂停从手术的CRC样本活体细胞的准备工作流程。 1。临床样本分类所有样品均采自皇家阿尔弗雷德王子医院（坎珀，NSW，澳大利亚）和协和遣返医院（协和西澳大利亚新南威尔士州）X08 - 164号议定书下的知情同意。 <ol><li>收集的新鲜大肠癌（CRC）的或腺瘤标本，和正常的肠道黏膜至少10...

<ol>
	<li>Steinert, R., Buschmann, T., vander Linden, M., Fels, L. M., Lippert, H., Reymond, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+proteomics+in+the+diagnosis+and+outcome+prediction+in+colorectal+cancer.">The role of proteomics in the diagnosis and outcome prediction in colorectal cancer.</a> Technol. Cancer. Res. Treat. 1, 297 (2002).</li><li>Eifel, P., Axelson, J. A., Costa, J., Crowley, J., Curran, W. J., Deshler, A., Fulton, S., Hendricks, C. B., Kemeny, M., Kornblith, A. B., Louis, T. A., Markman, M., Mayer, R., Roter, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=National+Institutes+of+Health+Consensus+Development+Conference+Statement:+adjuvant+therapy+for+breast+cancer.">National Institutes of Health Consensus Development Conference Statement: adjuvant therapy for breast cancer.</a> J. Natl. Canc. Inst. 93, 979 (2001).</li><li>Swerdlow, S. H., Campo, E., Harris, H. L., Jaffe, E. S., Pileri, S. A., Stein, H., Thiele, J., Vardiman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WHO+classification+of+tumour+of+haematopoietic+and+lymphoid+tissues.">WHO classification of tumour of haematopoietic and lymphoid tissues.</a> IARC WHO Classification of Tumours. 2, (2008).</li><li>Xiao, G. G., Recker, R. R., Deng, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+proteomics+and+cancer+biomarker+discovery.">Recent advances in proteomics and cancer biomarker discovery.</a> Clin. Med. Oncol. , (2008).</li><li>Belov, L., Mulligan, S. P., Barber, N., Woolfson, A., Scott, M., Stoner, K., Chrisp, J. S., Sewell, W. A., Bradstock, K. F., Bandall, L., Pascovici, D. S., Thomas, M., Erber, W., Huang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+human+leukaemias+and+lymphomas+using+extensive+immunophenotypes+from+an+antibody+microarray.">Analysis of human leukaemias and lymphomas using extensive immunophenotypes from an antibody microarray.</a> Br. J. Haematol. 135, 184 (2006).</li><li>Belov, L., Huang, P., Barber, N., Mulligan, S. P., Christopherson, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+repertories+of+surface+antigens+on+leukemias+using+an+antibody+microarray.">Identification of repertories of surface antigens on leukemias using an antibody microarray.</a> Proteomics. 3, 2147 (2003).</li><li>Zhou, J., Belov, L., Huang, P. Y., Shin, J., Solomon, M. J., Chapuis, P. H., Bokey, L., Chan, C., Clarke, C., Clarke, S. J., Christopherson, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+antigen+profiling+of+colorectal+cancer+using+antibody+microarrays+with+fluorescence+multiplexing.">Surface antigen profiling of colorectal cancer using antibody microarrays with fluorescence multiplexing.</a> J. Immunol. Methods. 355 (1-2), 40-51 (2010).</li><li>Ellmark, P., Belov, L., Huang, P., Lee, C. S., Solomon, M. J., Morgan, D. K., Christopherson, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+detection+of+surface+molecules+on+colorectal+cancers.">Multiplex detection of surface molecules on colorectal cancers.</a> Proteomics. 6, 1791 (2006).</li><li>Pearson, J. P., Allen, A., Hutton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheology+of+mucin.">Rheology of mucin.</a> Methods Mol. Biol. 125, 99 (2000).</li><li>Yang, Y. H., Dudoit, S., Luu, P., Lin, D. M., Peng, V., Ngai, J., Speed, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+for+cDNA+microarray+data:+a+robust+composite+method+addressing+single+and+multiple+slide+systematic+variation.">Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation.</a> Nucleic Acids Res. 30, 15 (2002).</li><li>Al Saeed, ., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TMA:+A+free,+open-source+system+for+microarray+data+management+and+analysis.">TMA: A free, open-source system for microarray data management and analysis.</a> BioTechniques. 34, 374-378 (2003).</li></ol>

在这段视频中，我们将演示如何DotScan抗体芯片可以在一个简单的，半定量的方法来研究从启联资源中心组织的细胞群表面抗原型材的使用。 从组织中获得一个可行的单细胞悬液，实验的成功是至关重要的，因为似乎是公司具有约束力的全细胞抗体点的孵化过程中所需的能源依赖的过程（如抗原上限和/或伪足形成），而随后冲关死细胞。 1型胶原酶最初受雇于组织disaggregation8?...

The author's email has been corrected in the publication of <a href="http://www.jove.com/video/3322">Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing</a>. There was an error with the author, Jerry Zhou's, email. The author's email has been updated to:
j.zhou@uws.edu.au
from:
jzho7551@mail.usyd.edu.au

Erratum: Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

<table border="1"><tbody><tr><th>试剂或设备名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>汉克斯“平衡盐溶液</td><td> Sigma - Aldrich公司</td><td> H6136 - 10X1L </td><td>缓冲25毫米HEPES（Sigma公司＃H3375） </td></tr><tr><td> Airpure生物安全柜II类</td><td>西屋电气公司</td><td> 1687-2340/612 </td><td></td></tr><tr><td>手术刀</td><td>利文斯通</td><td> 090609 </td><td>每包100 </td></tr><tr><td> 2毫米HEPES的RPMI 1640 </td><td> Sigma - Aldrich公司</td><td> R4130 - 10X1L </td><td></td></tr><tr><td>型胶原酶4 </td><td>沃辛顿</td><td> 4188 </td><td></td></tr><tr><td>脱氧核糖核酸1 </td><td> Sigma - Aldrich公司</td><td> DN25 - 1G </td><td></td></tr><tr><td> Terumo公司注射器（10毫升） </td><td> Terumo公司</td><td> SS 10大号</td><td>盒：100个</td></tr><tr><td> Filcon过滤器（200微米） </td><td> BD公司</td><td> 340615 </td><td></td></tr><tr><td> Filcon过滤器（50微米） </td><td> Filcon过滤器（50微米） </td><td> Filcon Filcon过滤器（50微米）过滤器（50微米）340603 </td><td></td></tr><tr><td>胎牛血清</td><td> GIBCO / Invitrogen公司</td><td> 10099-141 </td><td></td></tr><tr><td>离心5810 ř </td><td> Eppendorf公司</td><td> 7017 </td><td></td></tr><tr><td>二甲基亚砜</td><td> Sigma - Aldrich公司</td><td> D2650 </td><td></td></tr><tr><td>台盼蓝</td><td> Sigma - Aldrich公司</td><td> T8154 </td><td></td></tr><tr><td> Hemocymeter Technocolor Neubar </td><td>赫斯曼</td><td>不可用</td><td></td></tr><tr><td>光镜下</td><td>尼康</td><td>尼康TMS </td><td></td></tr><tr><td> Cyrovial管</td><td>格雷纳生物一</td><td> 121278 </td><td></td></tr><tr><td>低温冷冻contrainer </td><td> NALGENE </td><td> 5100-0001 </td><td></td></tr><tr><td> DotScan抗体芯片试剂盒</td><td> Medsaic </td><td>不可用</td><td></td></tr><tr><td> DotScan芯片洗托盘</td><td> Medsaic </td><td>不可用</td><td></td></tr><tr><td> KimWipes </td><td>金佰利</td><td> 4103 </td><td></td></tr><tr><td>甲醛37％ </td><td> Sigma - Aldrich公司</td><td> F1635 - 500ML </td><td></td></tr><tr><td> DotReaderTM </td><td> Medsaic </td><td>不可用</td><td></td></tr><tr><td>牛血清白蛋白</td><td> Sigma - Aldrich公司</td><td> A9418 - 10G </td><td></td></tr><tr><td>热灭活AB血清2％ </td><td> Invitrogen公司</td><td> 34005100 </td><td></td></tr><tr><td>藻红蛋白的共轭CD3 </td><td> Beckman Coulter公司</td><td> ET386 </td><td></td></tr><tr><td> AlexaFluor647 -共轭EpCAM的</td><td> BioLegend </td><td> 324212 </td><td></td></tr><tr><td>台风的FLA 9000 </td><td> GE医疗集团</td><td> 28-9558-08 </td><td> 532 nm激光，580 BP30 PE排放过滤器。 633 nm激光和670 BP30 Alexa647排放过滤器</td></tr><tr><td> MultiExperiment浏览器V4.4 </td><td> TM4芯片软件套件</td><td>打开 - 源码软件（参11） </td><td></td></tr></tbody></table>

colorectal cancer cell surface protein profiling using an antibody microarray and fluorescence multiplexing

依靠目前的预后和“儿童权利公约”的分类分期，病理组织学和临床研究结果结合起来的系统。然而，在大多数“儿童权利公约”的情况下，细胞功能障碍是众多的基因突变，修改蛋白表达和翻译后修饰1的结果。 一些细胞表面抗原，包括集群分化（CD）抗原，已被确定为潜在的预后或转移的生物标志物在CRC。这些抗原的表达理想的生物标志物往往与肿瘤进展或与其他类型的细胞，如互动的变化的肿瘤浸润淋巴细胞（TILS）和肿瘤相关的（TAMS）巨噬细胞。 使用免疫组织化学（IHC）癌症的子分类和预测是公认的对某些类型的肿瘤2,3。但是，没有一个“标记”显示预后意义大于临床病理分期或获得广泛接受，在所有儿童权利公约“情况下的常规病理报告为使用。 最近的方针疾病表型的预后分层依赖的表面蛋白的配置文件使用多个“标记”。虽然表达的肿瘤蛋白质组技术如iTRAQ分析是一个功能强大的工具biomarkers4发现，它不是诊断实验室常规使用的最佳选择，不能区分不同类型的细胞在混合人口。此外，肿瘤组织大量的纯化的质膜糖蛋白的分析，这些方法都需要。 在这段视频中，我们描述了一个简单的方法，从分类的CRC使用DotScan CRC抗体芯片的样品活细胞的表面蛋白质组分析。 122抗体芯片包括一个标准的82 -抗体确认系特定标记的白细胞粘附分子，受体和炎症和免疫反应 5标志物的范围，CRC的40个潜在的预后标志物的检测与卫星地区的区域。细胞被捕获，只对他们表达相应抗原的抗体。光学扫描确定，每个点的细胞密度，反映了细胞表达，抗原，抗原的表达和亲和力的抗体6水平的比例。 CRC的组织或正常的肠道黏膜，光学扫描反映免疫细胞混合种群。然后，可以使用复用荧光来分析细胞阵列上捕获的利益选择的细分人群。例如，Alexa的647抗上皮细胞粘附分子（EpCAM的; CD326），是一个泛上皮被用来检测的CRC细胞和正常的肠道黏膜上皮细胞的分化抗原，而藻红蛋白 - 抗CD3，检测浸润T细胞7。 DotScan的CRC芯片应诊断替代的解剖学基础CRC分期系统的原型。

Watch this Scientific Journal Video about Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing at JoVE.com

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

我们描述了一个过程分解大肠直肠癌（CRC）来产生可行的单个细胞，然后在定制的抗体，认识表面抗原（DotScan CRC芯片）芯片捕获。绑定到芯片的细胞亚群可以分析使用荧光标记的单克隆抗体与荧光染料的复用。

大肠癌细胞表面蛋白的分析，使用一种抗体的芯片和荧光多路复用

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

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