और मानव Glioblastoma Carboxy Fluorescein Succinimidyl एस्टर (CFSE) में धीमी गति से विभाजित कोशिकाओं की पहचान अलगाव
Summary
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.
Abstract
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.
Protocol
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Name of the reagent | Type | Company | Catalogue number |
| NeuroCult NSC Basal Medium (Human) | Medium | Stem Cell Technologies | 05750 |
| NeuroCult NSC Proliferation Supplements (Human) | Medium supplement | Stem Cell Technologies | 05753 |
| %0.05 trypsin-EDTA | Reagent | Gibco | 25300-062 |
| Soybean trypsin inhibitor | Reagent | Sigma | T6522 |
| Penicillin/Streptomycin | Reagent | Gibco | 15140-122 |
| T25 flask | Culture ware | Nalge Nunc international | 136196 |
| T80 flask | Culture ware | Nalge Nunc international | 178905 |
| 15 ml tubes | Culture ware | BD Falcon | 352096 |
| 50 ml tubes | Culture ware | BD Falcon | 352070 |
| EGF | Growth factor | R&D | 2028-EG |
| CellTrace CFSE Cell Proliferation Kit | Fluorescent Cell Division Tracking Dye | Molecular Probes, Invitrogen | C34554 |
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