Name: isolation of normal and cancer-associated fibroblasts from fresh tissues by fluorescence activated cell sorting (facs)
Uploaded: 2013-01-14T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS at JoVE.com

Vi takker Dr. Yitzchak Oschry og Dr. Orit Sagi-Asif for deres hjælp med FACS sortering. Denne forskning blev støttet af tilskud til NE fra Den Europæiske Union syvende rammeprogram (FP7/2007-2013) under tilskudsaftale nr. [276.890], fra Israel Cancer Association (# 20.110.078), og fra Israel Cancer Research Fund (Research Career Development Award).

1. Dissekering Mammary eller hudvæv fra mus <ol><li> Før dissekere det ønskede væv fra mus, forberede følgende leverancer og reagenser: Tilbehør: <ul><li> Kirurgiske værktøjer (vasket med detergent og derefter dyppet i 70% ethanol). </li><li> Styrofoam underlag til at placere musen på under dissektion. </li><li> Stifter eller nåle til at holde musene under dissektion. </li><li> Glaskrukke med låg for fordøjelsen, som indeholder magnetisk omrørerstav (autoklaveret). </li><li> Vand...

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Medens eksperimenter udført i vævskultur kan være informative og antyder funktionelle principper, der kan verificeres in vivo, er det kendt, at store ændringer i genekspression i celler i kultur 11,12. For at undgå en vævskultur trin ved profilering af genekspression i fibroblaster, har vi udviklet en protokol, der tillader isolering af normale, såvel som cancer-associerede fibroblaster fra friske mus eller humant væv. Denne protokol blev anvendt med succes med hud, pankreas og brystvæv fra ...

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isolation of normal and cancer-associated fibroblasts from fresh tissues by fluorescence activated cell sorting (facs)

Cancerassocierede fibroblaster (CAFS) er de mest fremtrædende celletype i tumorstroma af mange cancere, især brystcancer, og deres fremtrædende tilstedeværelse er ofte forbundet med dårlig prognose 1,2. CAFS er en aktiveret subpopulation af stromale fibroblaster, hvoraf mange udtrykker myofibroblast markør α-SMA 3. CAFS stammer fra lokale væv fibroblaster såvel som fra knoglemarv-afledte celler rekrutteres til det udviklende tumor og vedtage en CAF fænotype under indflydelse af den tumormikromiljøet 4. CAFS blev vist at lette tumorinitiering, vækst og progression gennem signalering som fremmer tumorcelledeling, angiogenese og invasion 5-8. Vi viste, at CAFS forbedre tumorvækst ved at formidle tumor-fremmende inflammation, der starter tidligst præneoplastiske trin 9. Trods stigende tegn på den vigtige rolle, CAFS spille ved at lette tumor vækst,studere CAFS har været en løbende udfordring på grund af manglen på CAF-specifikke markører og det store forskellighed disse celler, med mange undertyper sameksisterende i tumormikromiljøet 10. Endvidere undersøger fibroblaster in vitro hindres af, at deres genekspression nu ofte ændres i vævskultur 11,12. For at løse dette problem og muliggøre uvildig genekspression profilering af fibroblaster fra frisk mus og humane væv, vi udviklet en metode baseret på tidligere protokoller for Fluorescence cellesortering (FACS) 13,14. Vores fremgangsmåde er baseret på anvendelse af PDGFRα som overflademarkør at isolere fibroblaster fra frisk muse-og humant væv. PDGFRα er rigeligt udtrykt ved både normale fibroblaster og CAFS 9,15. Denne fremgangsmåde tillader isolering af rene populationer af normale fibroblaster og cafeer, herunder, men ikke begrænset til α-SMA + aktiverede myofibroblaster. Isolerede fibroblaster kan derefteranvendes til karakterisering og sammenligning af udviklingen i genekspression, der opstår i CAFS under tumorigenese. Faktisk vi og andre rapporterede udtryk profilering af fibroblaster isoleret ved cellesortering 16. Denne protokol blev med succes udført for at isolere og profilere højt beriget populationer af fibroblaster fra hud, bryst, bugspytkirtel og lungevæv. Desuden er vores fremgangsmåde tillader også dyrkning af sorterede celler, for at udføre funktionelle forsøg og for at undgå forurening med tumorceller, hvilket ofte er en stor hindring, når de forsøger at dyrke CAFS.

Ved hjælp PDGFRα som markør for fibroblaster resulterer i isolering af højt beriget populationer af væv fibroblaster. Det niveau af renhed efter sortering var 99%, som kvantificeret ved post-sort analyse (figur 2A). Skønne det procentiske kontaminerende ikke-fibroblastceller ved den relative ekspression af celle-specifikke kontrol-gener (figur 2B) viser typisk 0,1-0,6% kontaminering. Dette niveau af renhed tillader høj kvalitet transkriptom profilering af isolerede fibroblaster <...

Watch this Scientific Journal Video about Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS at JoVE.com

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer associeret Fibroblaster (CAFS) lette tumor initiering, vækst og progression gennem signalere, der fremmer spredning, angiogenese og inflammation. Her beskriver vi en fremgangsmåde til at isolere rene populationer af normale fibroblaster og CAFS fra frisk muse og humane væv ved cellesortering under anvendelse PDGFRα som overflademarkør.

Isolering af normale og cancer-associeret fibroblaster fra friske væv ved fluorescens-aktiveret cellesortering (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

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Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

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During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

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