Document Detail


Identification and isolation of slow-dividing cells in human glioblastoma using carboxy fluorescein succinimidyl ester (CFSE).
MedLine Citation:
PMID:  22565048     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies(1). 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 leukemia(2-8). 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 glioblastoma(2,7,8). 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 bonds(9). 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 division(10). 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 activity(2). 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 cytometry(2). 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.
Authors:
Loic P Deleyrolle; Mark R Rohaus; Jeff M Fortin; Brent A Reynolds; Hassan Azari
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Video-Audio Media     Date:  2012-04-29
Journal Detail:
Title:  Journal of visualized experiments : JoVE     Volume:  -     ISSN:  1940-087X     ISO Abbreviation:  J Vis Exp     Publication Date:  2012  
Date Detail:
Created Date:  2012-05-08     Completed Date:  2012-07-20     Revised Date:  2014-05-12    
Medline Journal Info:
Nlm Unique ID:  101313252     Medline TA:  J Vis Exp     Country:  United States    
Other Details:
Languages:  eng     Pagination:  -     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Brain Neoplasms / pathology*
Cell Growth Processes / physiology
Flow Cytometry / methods*
Fluoresceins / chemistry*
Fluorescent Dyes / chemistry*
Glioblastoma / pathology*
Humans
Staining and Labeling / methods*
Succinimides / chemistry*
Tumor Cells, Cultured
Grant Support
ID/Acronym/Agency:
1R21CA141020-01/CA/NCI NIH HHS
Chemical
Reg. No./Substance:
0/5-(6)-carboxyfluorescein diacetate succinimidyl ester; 0/Fluoresceins; 0/Fluorescent Dyes; 0/Succinimides
Comments/Corrections

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