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

Мы благодарим сотрудников Анатомические лаборатории патологии Королевский Принца Альфреда и Согласия Репатриация Больницы для сбора свежих образцов КПР и нормальной слизистой оболочки кишечника. Работа финансировалась института рака Нового Южного Уэльса Поступательное Программа грантов.

<img src="/files/ftp_upload/3322/3322fig1.jpg" alt="Рисунок 1" /> Рисунок 1. Набор операций для приготовления суспензии живых клеток от хирургического образца о правах ребенка. 1. Клинический пример разбивки Все образцы были собраны из Королевской больницы Принца Альфр...

<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>

В этом видео показано, как DotScan микрочипов антитела могут быть использованы в простых, полуколичественный способ изучения профилей поверхностного антигена для популяции клеток из ткани КПР. Получение жизнеспособных суспензии отдельных клеток из ткани имеет решающее з...

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> Название реагентов или оборудования </th><th> Компания </th><th> Номер в каталоге </th><th> Комментарии </th></tr><tr><td> Сбалансированный солевой Хэнкса решение </td><td> Sigma-Aldrich </td><td> H6136-10X1L </td><td> Буферизацией с 25 мМ Hepes (Sigma # H3375) </td></tr><tr><td> Airpure биологической безопасности II класса кабинет </td><td> Westinghouse </td><td> 1687-2340/612 </td><td></td></tr><tr><td> Хирургические лезвия </td><td> Ливингстон </td><td> 090609 </td><td> Упаковка из 100 </td></tr><tr><td> RPMI 1640 с 2 мМ Hepes </td><td> Sigma-Aldrich </td><td> R4130-10X1L </td><td></td></tr><tr><td> Коллагеназы типа 4 </td><td> Уортингтон </td><td> 4188 </td><td></td></tr><tr><td> Дезоксирибонуклеазы 1 </td><td> Sigma-Aldrich </td><td> DN25-1G </td><td></td></tr><tr><td> Terumo шприц (10 мл) </td><td> Terumo </td><td> SS +10 L </td><td> Комплект из 100 </td></tr><tr><td> Filcon фильтр (200 мкм) </td><td> BD Biosciences </td><td> 340615 </td><td></td></tr><tr><td> Filcon фильтр (50 мкм) </td><td> Filcon фильтр (50 мкм) </td><td> Filcon фильтр (50 мкм) Filcon фильтр (50 мкм) 340603 </td><td></td></tr><tr><td> Фетальной телячьей сыворотки </td><td> Gibco / Invitrogen </td><td> 10099-141 </td><td></td></tr><tr><td> Центрифуга 5810 R </td><td> Эппендорф </td><td> 7017 </td><td></td></tr><tr><td> Диметилсульфоксид </td><td> Sigma-Aldrich </td><td> D2650 </td><td></td></tr><tr><td> Трипановый синий </td><td> Sigma-Aldrich </td><td> T8154 </td><td></td></tr><tr><td> Hemocymeter Technocolor Neubar </td><td> Hirschmann </td><td> недоступный </td><td></td></tr><tr><td> Оптический микроскоп </td><td> Nikon </td><td> Nikon TMS </td><td></td></tr><tr><td> Cyrovial труб </td><td> Greiner Bio-One </td><td> 121278 </td><td></td></tr><tr><td> Криогенная заморозка contrainer </td><td> Nalgene </td><td> 5100-0001 </td><td></td></tr><tr><td> DotScan антител микрочипов комплект </td><td> Medsaic </td><td> недоступный </td><td></td></tr><tr><td> DotScan микрочипов мыть лоток </td><td> Medsaic </td><td> недоступный </td><td></td></tr><tr><td> KimWipes </td><td> Kimberly-Clark </td><td> 4103 </td><td></td></tr><tr><td> Формальдегид 37% </td><td> Sigma-Aldrich </td><td> F1635-500ML </td><td></td></tr><tr><td> DotReaderTM </td><td> Medsaic </td><td> недоступный </td><td></td></tr><tr><td> Бычий сывороточный альбумин </td><td> Sigma-Aldrich </td><td> A9418-10G </td><td></td></tr><tr><td> Тепло-инактивированной сывороткой AB 2% </td><td> Invitrogen </td><td> 34005100 </td><td></td></tr><tr><td> Фикоэритрин-сопряженных CD3 </td><td> Beckman Coulter </td><td> ET386 </td><td></td></tr><tr><td> AlexaFluor647-сопряженных EpCam </td><td> BioLegend </td><td> 324212 </td><td></td></tr><tr><td> Тайфун FLA-9000 </td><td> GE Healthcare </td><td> 28-9558-08 </td><td> 532 нм, 580 BP30 выбросов фильтр для PE. 633 лазерных нм и 670 BP30 выбросов фильтр для Alexa647 </td></tr><tr><td> MultiExperiment просмотра v4.4 </td><td> ТМ4 Microarray Software Suite </td><td> Открыть - программное обеспечение (Ссылка 11) </td><td></td></tr></tbody></table>

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

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority of CRC cases, cell dysfunction is the result of numerous mutations that modify protein expression and post-translational modification1.
A number of cell surface antigens, including cluster of differentiation (CD) antigens, have been identified as potential prognostic or metastatic biomarkers in CRC. These antigens make ideal biomarkers as their expression often changes with tumour progression or interactions with other cell types, such as tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs).
The use of immunohistochemistry (IHC) for cancer sub-classification and prognostication is well established for some tumour types2,3. However, no single &#x2018;marker&#x2019; has shown prognostic significance greater than clinico-pathological staging or gained wide acceptance for use in routine pathology reporting of all CRC cases. 
A more recent approach to prognostic stratification of disease phenotypes relies on surface protein profiles using multiple 'markers'. While expression profiling of tumours using proteomic techniques such as iTRAQ is a powerful tool for the discovery of biomarkers4, it is not optimal for routine use in diagnostic laboratories and cannot distinguish different cell types in a mixed population. In addition, large amounts of tumour tissue are required for the profiling of purified plasma membrane glycoproteins by these methods. 
In this video we described a simple method for surface proteome profiling of viable cells from disaggregated CRC samples using a DotScan CRC antibody microarray. The 122-antibody microarray consists of a standard 82-antibody region recognizing a range of lineage-specific leukocyte markers, adhesion molecules, receptors and markers of inflammation and immune response5, together with a satellite region for detection of 40 potentially prognostic markers for CRC. Cells are captured only on antibodies for which they express the corresponding antigen. The cell density per dot, determined by optical scanning, reflects the proportion of cells expressing that antigen, the level of expression of the antigen and affinity of the antibody6. 
For CRC tissue or normal intestinal mucosa, optical scans reflect the immunophenotype of mixed populations of cells. Fluorescence multiplexing can then be used to profile selected sub-populations of cells of interest captured on the array. For example, Alexa 647-anti-epithelial cell adhesion molecule (EpCAM; CD326), is a pan-epithelial differentiation antigen that was used to detect CRC cells and also epithelial cells of normal intestinal mucosa, while Phycoerythrin-anti-CD3, was used to detect infiltrating T-cells7. The DotScan CRC microarray should be the prototype for a diagnostic alternative to the anatomically-based CRC staging system.

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

Мы описали процедуру разбивки колоректального рака (КРР) для получения жизнеспособных отдельных клеток, которые затем захвачен на индивидуальные микрочипов антител признании поверхностных антигенов (DotScan CRC микрочипов). Суб-популяции клеток, связанных с микрочипов можно профилировать по флуоресценции мультиплексирования с использованием моноклональных антител помеченные флуоресцентными красителями.

Колоректального рака на поверхности клеток белков профилировки с Microarray антитела и флуоресценции мультиплексирования

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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