Name: isolation and culture of neural crest cells from embryonic murine neural tube
Uploaded: 2012-06-02T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube at JoVE.com

Vi vil gerne anerkende Marc Wozniak til video assistance. Vi vil også gerne takke Sean Morrison på UT Southwestern for den oprindelige protokol for dyrkning af rotte NC celler. Dette arbejde blev støttet Vanderbilt University Medical Center Akademisk Program Support og ved tilskud fra NIH (HD36720 og HD036720-11S109) og AHA 11GRNT7690040 til PAL, predoctoral stipendier fra AHA (0615209B) og NIH (NS065604) til NAM, og ERP var understøttet af en NIH træning tilskud T32HD007502.

1. Forbereder Plader <ol><li> Anvendelse af steril teknik til enhver tid. </li><li> Forberede fibronectin (FN) ved at fortynde 100 pi humant plasma FN lager til et slutvolumen på 3,3 ml i Dulbeccos PBS (DPBS). Endelige koncentration 30 ug / ml, og dette kan opbevares ved 4 ° C i 1 uge. </li><li> Omfatter bunden af ​​hver brønd i en steril vævskulturplade fire brønde med FN-opløsning og lad henstå i 15 minutter. Sørg for, at hele overfladen er dækket. Gør mediet i denne periode (trin 2 og 3). </li><li> Fj...

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Omhyggelig bør man være opmærksom på udviklingsstadiet af embryo for at sikre succes for denne tilgang. Optælling somitter af tidlige museembryoer er kritisk for både fase matche embryoner inden for et kuld hvalpe og bestemme de korrekte områder af neuralrøret for isolation. En variation af en eller to somitter mellem embryoer er inden for et rimeligt udvalg af udviklingsmæssige timing, afhængigt af opløsningen af ​​den gennemførte forsøget. Et embryo mellem 9 og 9,5 DPC vil have mellem 17 og 25 somitte...

<table border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> DMEM (lav glucose) </td><td> Gibco / Invitrogen </td><td> 11885 </td><td></td></tr><tr><td> Neurobasal Medium </td><td> Gibco </td><td> 21103 </td><td></td></tr><tr><td> BSA </td><td> Sigma </td><td> A3912-10G </td><td></td></tr><tr><td> DPBS </td><td> Gibco </td><td> 14190-144 </td><td></td></tr><tr><td> IGF1 </td><td> BD Biosciences </td><td> 354037 </td><td> Opbevares i 50 ug / ml alikvoter ved -20 ° C. </td></tr><tr><td> bFGF </td><td> BD Biosciences </td><td> 354060 </td><td> Opbevares i 25 ug / ml alikvoter ved -20 ° C. </td></tr><tr><td> Fibronektin </td><td> Gibco </td><td> 33016-015 </td><td> Lagret i 1mg/ml aliquots ved -20 ° C. </td></tr><tr><td> Retinsyre </td><td> Sigma </td><td> R2625 </td><td> Opbevares i 35 ug / ml aliquoter efter rekonstituering i ethanol ved -20 ° C. </td></tr><tr><td> 2-mercaptoethanol </td><td> Sigma </td><td> D-5637 </td><td></td></tr><tr><td> N2 supplement </td><td> Gibco </td><td> 17502-048 </td><td></td></tr><tr><td> B27 supplement </td><td> Gibco </td><td> 17504-044 </td><td></td></tr><tr><td> Steriflip 0,22 um filtre </td><td> Millipore </td><td> SCGP00525 </td><td></td></tr><tr><td> Penicillin-streptomycin </td><td> Invitrogen </td><td> 15140122 </td><td></td></tr><tr><td> 0,20 um filtre </td><td> Corning </td><td> 431219 </td><td></td></tr><tr><td> Sprøjter (til filtrering) </td><td> BD Biosciences </td><td> 301604 </td><td></td></tr&gt; <tr><td> Fire brønde </td><td> Thermo Fisher Scientific </td><td> 176740 </td><td></td></tr><tr><td> Collagenase / dispase </td><td> Roche </td><td> 269 ​​638 </td><td> Aktivitet varierer fra parti. Opbevares i 100 mg / ml alikvoter ved -20 ° C. </td></tr><tr><td> Insulin nåle (29 ½ Gage) </td><td> Becton Dickson </td><td> 309306 </td><td></td></tr><tr><td> Hypoxi Afdeling </td><td> Billups-Rothenberg </td><td></td><td></td></tr><tr><td> Oxygen Analyzer </td><td> Billups-Rothenberg </td><td></td><td></td></tr><tr><td> Tang # 5 </td><td> Gode ​​Science Tools </td><td></td><td> Til fjernelse uterus og decidua. </td></tr><tr><td> Trypsin-EDTA (0,25%) </td><td> Gibco </td><td> 25200 </td><td></td></tr></tbody></table>

isolation and culture of neural crest cells from embryonic murine neural tube

Den embryoniske crista neuralis (NC) er en multipotent progenitorcelle population, der stammer fra den dorsale aspekt af neuralrøret, undergår en epitelcelle til mesenchymal overgang (EMT) og migrerer gennem embryo, hvilket giver anledning til forskellige celletyper 1-3. NC har også den unikke evne til at påvirke differentiering og modning af målorganer 4-6. Når eksplanteret in vitro, NC progenitorer undergår selvfornyelse, migrerer og differentierer til en række forskellige vævstyper, herunder neuroner og glia, glatte muskelceller, brusk og knogler. NC multipotency blev først beskrevet af eksplantater af aviær neuralrøret 7-9. In vitro isolering af NC-celler letter undersøgelsen af NC dynamik, herunder proliferation, migration og multipotency. Det videre arbejde i de fugle og rotter systemer viste, at eksplanterede NC-celler bevarer deres NC potentiale, når transplanteres tilbage til fosteret 10-13. Fordi disse iboende cellulære egenskaber er bevaret i eksplanterede NC stamfædre, neuralrøret explantet analysen forsyner en attraktiv mulighed for at studere NC in vitro. At opnå en bedre forståelse af den mammale NC, er mange metoder blevet anvendt til at isolere NC populationer. NC-afledte progenitorceller kan dyrkes fra post-migrerende steder i både embryo og voksne at studere dynamikken af post-migrerende NC progenitorer 11,14-20 imidlertid isolering af NC progenitorer, da de emigrere fra neuralrøret tilvejebringer optimal konservering af NC celle potentiale og migrerende egenskaber 13,21,22. Nogle protokoller anvender fluorescensaktiveret cellesortering (FACS) for at isolere en NC population er beriget for bestemte progenitorer 11,13,14,17. Imidlertid, når der startes med tidlige embryoer, celleantal passende for analyser er vanskeligt at opnå med FACS, komplicerer isolering af tidlige NC populations fra individuelle embryoer. Her beskriver vi en tilgang, som ikke er afhængig af FACS og resultater i en ca 96% ren NC befolkning baseret på en Wnt1-Cre aktiveret slægt reporter 23. Fremgangsmåden fremlagt her, er tilpasset fra protokoller optimeret til dyrkning af rotte-NC 11,13. Fordelene ved denne protokol i forhold til tidligere metoder er, at 1) at cellerne ikke dyrkes på et fødelag, 2) FACS ikke nødvendig for at opnå en relativt ren population NC, 3) premigratory NC celler isoleres og 4) resultaterne er let kvantificeret. Endvidere kan denne protokol kan anvendes til isolering af NC fra enhver mutant musemodel, letter undersøgelsen af ​​NC egenskaber med forskellige genetiske manipulationer. Begrænsning ved denne fremgangsmåde er, at NC fjernes fra rammerne af embryo, som vides at påvirke overlevelse, migration og differentiering af NC 2,24-28.

Watch this Scientific Journal Video about Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube at JoVE.com

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

Isolering af embryoniske crista neuralis fra neuralrøret letter anvendelsen af<em&gt; In vitro</em&gt; Metoder til at studere migration, selvfornyelse, og multipotency af neural crest.

Isolering og dyrkning af neurale kamceller fra embryonale Murin Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

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