Name: identification and isolation of slow-dividing cells in human glioblastoma using carboxy fluorescein succinimidyl ester (cfse)
Uploaded: 2012-04-29T14:00:00
Description: Watch this Scientific Journal Video about Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE at JoVE.com

इस काम के ब्रेन ट्यूमर अनुसंधान, प्रेस्टन ए ब्रेन ट्यूमर चिकित्सा और स्वास्थ्य के राष्ट्रीय संस्थानों (1R21CA141020-01) के लिए वेल्स जूनियर केंद्र के लिए फ्लोरिडा केंद्र द्वारा समर्थित किया गया.

1. Glioblastoma एकल कक्ष निलंबन की तैयारी <ol><li> Gliomasphere संस्कृति की स्थापना की है और 2,11,12 पहले से वर्णित neurosphere परख (एनएसए) का उपयोग कर बनाए रखा. </li><li> Passaging gliomaspheres के लिए उपयुक्त समय क्षेत्रों से युक्त मध्यम हटा दिया है, एक ?...

<ol>
	<li>Tian, T., Olson, S., Whitacre, J. M., Harding, 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+origins+of+cancer+robustness+and+evolvability.">The origins of cancer robustness and evolvability.</a> Integr. Biol. (Camb). 3, 17-30 (2011).</li><li>Deleyrolle, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+label-retaining+tumour-initiating+cells+in+human+glioblastoma.">Evidence for label-retaining tumour-initiating cells in human glioblastoma.</a> Brain. 134 (Pt. 5), 1331-1343 (2011).</li><li>Krishnamurthy, K., Wang, G., Rokhfeld, D., Bieberich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deoxycholate+promotes+survival+of+breast+cancer+cells+by+reducing+the+level+of+pro-apoptotic+ceramide.">Deoxycholate promotes survival of breast cancer cells by reducing the level of pro-apoptotic ceramide.</a> Breast Cancer Res. 10, 106-106 (2008).</li><li>Pece, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+and+molecular+heterogeneity+of+breast+cancers+correlates+with+their+cancer+stem+cell+content.">Biological and molecular heterogeneity of breast cancers correlates with their cancer stem cell content.</a> Cell. 140, 62-73 (2010).</li><li>Roesch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+temporarily+distinct+subpopulation+of+slow-cycling+melanoma+cells+is+required+for+continuous+tumor+growth.">A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth.</a> Cell. 141, 583-594 (2010).</li><li>Dembinski, J. L., Krauss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+functional+analysis+of+a+slow+cycling+stem+cell-like+subpopulation+in+pancreas+adenocarcinoma.">Characterization and functional analysis of a slow cycling stem cell-like subpopulation in pancreas adenocarcinoma.</a> Clin. Exp. Metastasis. 26, 611-623 (2009).</li><li>Graham, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primitive,+quiescent,+Philadelphia-positive+stem+cells+from+patients+with+chronic+myeloid+leukemia+are+insensitive+to+STI571+in+vitro.">Primitive, quiescent, Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro.</a> Blood. 99, 319-325 (2002).</li><li>Holyoake, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primitive+quiescent+leukemic+cells+from+patients+with+chronic+myeloid+leukemia+spontaneously+initiate+factor-independent+growth+in+vitro+in+association+with+up-regulation+of+expression+of+interleukin-3.">Primitive quiescent leukemic cells from patients with chronic myeloid leukemia spontaneously initiate factor-independent growth in vitro in association with up-regulation of expression of interleukin-3.</a> Blood. 97, 720-728 (2001).</li><li>Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+dyes+for+lymphocyte+migration+and+proliferation+studies.">Fluorescent dyes for lymphocyte migration and proliferation studies.</a> Immunol. Cell Biol. 77, 499-508 (1999).</li><li>Lyons, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysing+cell+division+in+vivo+and+in+vitro+using+flow+cytometric+measurement+of+CFSE+dye+dilution.">Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution.</a> J. Immunol. Methods. 243, 147-154 (2000).</li><li>Azari, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Expansion+of+Human+Glioblastoma+Multiforme+Tumor+Cells+Using+the+Neurosphere+Assay.">Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay.</a> J. Vis. Exp. (56), e3633-e3633 (2011).</li><li>Azari, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Expansion+of+the+Adult+Mouse+Neural+Stem+Cells+Using+the+Neurosphere+Assay.">Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay.</a> J. Vis. Exp. (45), e2393-e2393 (2010).</li></ol>

पढ़ाई की बढ़ती संख्या एक धीमी गति से विभाजित कैंसर में कोशिकाओं जो ट्यूमर गठन और उपचार 2-8 प्रतिरोध योगदान की उप डिब्बे के महत्व का प्रदर्शन किया है. इसलिए यह वर्णन करने के लिए और प्रयोगात्मक कोशिका?...

<table border="1"><tbody><tr><td> अभिकर्मक के नाम </td><td> टाइप </td><td> कंपनी </td><td> सूचीपत्र संख्या </td></tr><tr><td> NeuroCult एनएससी बेसल मध्यम (मानव) </td><td> मध्यम </td><td> स्टेम सेल टेक्नोलॉजीज </td><td> 05750 </td></tr><tr><td> NeuroCult एनएससी प्रसार की खुराक (मानव) </td><td> मध्यम पूरक </td><td> स्टेम सेल टेक्नोलॉजीज </td><td> 05753 </td></tr><tr><td> % 0.05 trypsin EDTA </td><td> अभिकर्मक </td><td> Gibco </td><td> 25300-062 </td></tr><tr><td> सोयाबीन trypsin अवरोध करनेवाला </td><td> अभिकर्मक </td><td> सिग्मा </td><td> T6522 </td></tr><tr><td> पेनिसिलिन स्ट्रेप्टोमाइसिन / </td><td> अभिकर्मक </td><td> Gibco </td><td> 15140-122 </td></tr><tr><td> T25 फ्लास्क </td><td> संस्कृति के बर्तन </td><td> Nalge Nunc अंतरराष्ट्रीय </td><td> 136196 </td></tr><tr><td> T80 फ्लास्क </td><td> संस्कृति के बर्तन </td><td> Nalge Nunc अंतरराष्ट्रीय </td><td> 178905 </td></tr><tr><td> 15 मिलीलीटर ट्यूब </td><td> संस्कृति के बर्तन </td><td> BD फाल्कन </td><td> 352096 </td></tr><tr><td> 50 मिलीलीटर ट्यूब </td><td> संस्कृति के बर्तन </td><td> BD फाल्कन </td><td> 352070 </td></tr><tr><td> EGF </td><td> वृद्धि कारक </td><td> अनुसंधान एवं विकास </td><td> २०२८ - जैसे </td></tr><tr><td> CellTrace CFSE सेल प्रसार किट </td><td> फ्लोरोसेंट कोशिका विभाजन डाई ट्रैकिंग </td><td> आणविक जांच, Invitrogen </td><td> C34554 </td></tr></tbody></table>

identification and isolation of slow-dividing cells in human glioblastoma using carboxy fluorescein succinimidyl ester (cfse)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Watch this Scientific Journal Video about Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE at JoVE.com

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

इस वीडियो प्रोटोकॉल फ्लोरोसेंट डाई carboxyfluorescein succinimidyl एस्टर (CFSE) की पहचान और मानव glioblastoma में कोशिकाओं के विभिन्न उप आबादी कोशिका विभाजन की आवृत्ति के आधार पर अलग होने के लिए आवेदन को दर्शाता है.

और मानव Glioblastoma Carboxy Fluorescein Succinimidyl एस्टर (CFSE) में धीमी गति से विभाजित कोशिकाओं की पहचान अलगाव

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

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Medicine

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

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