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

हम रॉयल प्रिंस अल्फ्रेड और Concord प्रत्यावर्तन अस्पताल सीआरसी और सामान्य आंत्र mucosa की ताजा नमूनों को इकट्ठा करने के लिए शारीरिक पैथोलॉजी प्रयोगशालाओं में स्टाफ धन्यवाद. काम कैंसर संस्थान के न्यू साउथ वेल्स translational कार्यक्रम अनुदान द्वारा वित्त पोषित किया गया था.

<img src="/files/ftp_upload/3322/3322fig1.jpg" alt="चित्रा 1" /> चित्रा 1. सीआरसी की एक शल्य नमूना से जीवित कोशिकाओं के निलंबन की तैयारी के लिए कार्य प्रवाह . 1. क्लीनिकल नमूना disaggregation प्रोटोकॉल X08 164 सं के तहत सूचि?...

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	<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>

इस वीडियो में, हम का प्रदर्शन कैसे DotScan एंटीबॉडी माइक्रोएरे एक सरल, अर्द्ध मात्रात्मक सीआरसी ऊतकों से सेल आबादी के लिए सतह प्रतिजन प्रोफाइल का अध्ययन में इस्तेमाल किया जा सकता है. ऊतक से एक व्?...

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> अभिकर्मक या उपकरण के नाम </th><th> कंपनी </th><th> सूचीपत्र संख्या </th><th> टिप्पणियाँ </th></tr><tr><td> हैंक्स &#39;संतुलित नमक समाधान </td><td> सिग्मा Aldrich </td><td> H6136-10X1L </td><td> 25 मिमी Hepes (# सिग्मा H3375) के साथ buffered </td></tr><tr><td> Airpure जैविक सुरक्षा कैबिनेट वर्ग द्वितीय </td><td> वेस्टिंगहाउस </td><td> 1687-2340/612 </td><td></td></tr><tr><td> सर्जिकल ब्लेड </td><td> लिविंग्स्टोन </td><td> 090609 </td><td> 100 के पैक </td></tr><tr><td> 2 मिमी Hepes के साथ 1640 RPMI </td><td> सिग्मा Aldrich </td><td> R4130-10X1L </td><td></td></tr><tr><td> Collagenase 4 </td><td> वर्थिंगटन </td><td> 4188 </td><td></td></tr><tr><td> 1 Deoxyribonuclease </td><td> सिग्मा Aldrich </td><td> DN25-1G </td><td></td></tr><tr><td> Terumo सिरिंज (10 एमएल) </td><td> Terumo </td><td> एसएस 10 एल </td><td> 100 के बॉक्स </td></tr><tr><td> Filcon फिल्टर (200 सुक्ष्ममापी) </td><td> बी.डी. बायोसाइंसेज </td><td> 340615 </td><td></td></tr><tr><td> Filcon फिल्टर (50 सुक्ष्ममापी) </td><td> Filcon फिल्टर (50 सुक्ष्ममापी) </td><td> Filcon फ़िल्टर (50 सुक्ष्ममापी) Filcon फिल्टर (50 सुक्ष्ममापी) 340603 </td><td></td></tr><tr><td> भ्रूण बछड़ा सीरम </td><td> / Gibco Invitrogen </td><td> 10099-141 </td><td></td></tr><tr><td> 5810 आर अपकेंद्रित्र </td><td> Eppendorf </td><td> 7017 </td><td></td></tr><tr><td> डाइमिथाइल sulphoxide </td><td> सिग्मा Aldrich </td><td> D2650 </td><td></td></tr><tr><td> Trypan नीले </td><td> सिग्मा Aldrich </td><td> T8154 </td><td></td></tr><tr><td> Hemocymeter Technocolor Neubar </td><td> HIRSCHMANN </td><td> अनुपलब्ध </td><td></td></tr><tr><td> प्रकाश माइक्रोस्कोप </td><td> Nikon </td><td> Nikon टीएमएस </td><td></td></tr><tr><td> Cyrovial ट्यूबों </td><td> Greiner जैव - एक </td><td> 121278 </td><td></td></tr><tr><td> क्रायो ठंड contrainer </td><td> Nalgene </td><td> 5100-0001 </td><td></td></tr><tr><td> DotScan एंटीबॉडी माइक्रोएरे किट </td><td> Medsaic </td><td> अनुपलब्ध </td><td></td></tr><tr><td> DotScan माइक्रोएरे धोने ट्रे </td><td> Medsaic </td><td> अनुपलब्ध </td><td></td></tr><tr><td> KimWipes </td><td> Kimberly-क्लार्क </td><td> 4103 </td><td></td></tr><tr><td> Formaldehyde 37% </td><td> सिग्मा Aldrich </td><td> F1635-500ml </td><td></td></tr><tr><td> DotReaderTM </td><td> Medsaic </td><td> अनुपलब्ध </td><td></td></tr><tr><td> गोजातीय सीरम albumin </td><td> सिग्मा Aldrich </td><td> A9418-10G </td><td></td></tr><tr><td> हीट निष्क्रिय अटल बिहारी सीरम 2% </td><td> Invitrogen </td><td> 34005100 </td><td></td></tr><tr><td> Phycoerythrin - संयुग्मित CD3 </td><td> Beckman कल्टर </td><td> ET386 </td><td></td></tr><tr><td> AlexaFluor647 - संयुग्मित EpCAM </td><td> BioLegend </td><td> 324212 </td><td></td></tr><tr><td> आंधी FLA 9000 </td><td> जीई हेल्थकेयर </td><td> 28-9558-08 </td><td> 532 एनएम लेजर, 580 पीई के लिए BP30 उत्सर्जन फिल्टर. 633 एनएम लेजर और 670 Alexa647 लिए BP30 उत्सर्जन फिल्टर </td></tr><tr><td> MultiExperiment v4.4 व्यूअर </td><td> TM4 माइक्रोएरे सॉफ्टवेयर सुइट </td><td> ओपन - स्रोत सॉफ्टवेयर (11 रेफरी) </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

हम कोलोरेक्टल कैंसर (सीआरसी) के लिए व्यवहार्य एकल कक्षों, जो तब अनुकूलित सतह एंटीजन (DotScan सीआरसी माइक्रोएरे) को पहचानने एंटीबॉडी प्रोटीन पर कब्जा कर रहे हैं उत्पादन के disaggregation के लिए एक प्रक्रिया का वर्णन. माइक्रोएरे के लिए बाध्य कोशिकाओं के उप - आबादी प्रतिदीप्ति फ्लोरोसेंट रंजक के साथ टैग मोनोक्लोनल एंटीबॉडी का उपयोग बहुसंकेतन द्वारा profiled किया जा सकता है है.

कोलोरेक्टल कैंसर कोशिका की सतह प्रोटीन रूपरेखा का प्रयोग एक एंटीबॉडी माइक्रोएरे और प्रतिदीप्ति बहुसंकेतन

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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