Name: dissection and culture of chick statoacoustic ganglion and spinal cord explants in collagen gels for neurite outgrowth assays
Uploaded: 2011-12-20T13:00:00
Description: Watch this Scientific Journal Video about Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays at JoVE.com

Dette arbejde blev finansieret af National Institutes of Health Grant RO1DC002756 og Purdue Research Foundation. Vi takker Doris Wu og Wiese Chang for rådgivning med eksperimenter og Rodney McPhail for at få hjælp med tal.

Opskrifter Chick Ringers <table border="1"><tbody><tr><td> 7,2 g </td><td> NaCl </td></tr><tr><td> 0,23 g </td><td> CaCl 2 + 2H 2 O </td></tr><tr><td> 0,37 g </td><td> KCl </td></tr><tr><td> 0,115 g </td><td> Na 2 HPO 4 </td></tr><tr><td> 900 ml </td><td> Vand (Vævskultur klasse) </td></tr></tbody></table> Bemærk: Justér pH til 7,4. Bring endelige volumen til 1000 ml med vand. 10X PBS <tabl...

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Vi præsenterer en metode til at dissekere og kultur E4 SAG og E6 rygmarven eksplanteret, fra chick, under serum-fri betingelser. Denne procedure er i øjeblikket anvendes i vores laboratorium for at studere effekten af ​​forskellige udskilles ligander på SAG neuron overlevelse og neurite udvækst. Nye aspekter af denne protokol omfatter brugen af ​​et serum-fri system til dyrkning eksplanteret og brug af perler vædet i vækstfaktorer for at studere virkningerne på SAG neurite udvækst. En perle metode magen ...

<table border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Katalog nummer </td><td> Kommentar </td></tr><tr><td> Udstyr </td><td></td><td></td><td></td></tr><tr><td> Dissektion mikroskop </td><td></td><td></td><td> Vi bruger en Leica M80 stereomikroskop med brightfield basen belysning </td></tr><tr><td> Slide varmere </td><td> CS &amp; E. </td><td></td><td> Kollagen polymerisering </td></tr><tr><td> Kylling æg inkubator </td><td></td><td></td><td></td></tr><tr><td> 37 ° C / 5% CO 2 befugtet cellekultur inkubator </td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Dissektion Materialer </td><td></td><td></td><td></td></tr><tr><td> Sylgard &amp;kopi belagt petriskål </td><td></td><td></td><td></td></tr><tr><td> 24-godt cellekultur plade </td><td> Corning </td><td> 3526 </td><td></td></tr><tr><td> # 5 Dumont pincet </td><td> Fin Science Tools </td><td> 11252-30 </td><td></td></tr><tr><td> # 55 Dumont pincet </td><td> Fin Science Tools </td><td> 11295-51 </td><td></td></tr><tr><td> Rustfrit stål dissekere stifter </td><td> Fin Science Tools </td><td> 26002-10 </td><td> Embryo pinning, fine dissektion </td></tr><tr><td> Moria perforeret skeen </td><td> Fin Science Tools </td><td> 10370-17 </td><td> Embryo høst / overførsel </td></tr><tr><td> 200 μl bred mund pipettespidser </td><td> Dot Scientific Inc </td><td> 118-96R </td><td> Eksplantation transfer </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> E4 og E6 hønseæg </td><td></td><td></td><td> &amp; Nbsp; </td></tr><tr><td> Chick Ringers opløsning </td><td></td><td></td><td> Embryo høst </td></tr><tr><td> HBSS </td><td> Sigma </td><td> H8264 </td><td> SAG dissektion </td></tr><tr><td> L15 medium </td><td> Sigma </td><td> L5520 </td><td> Rygmarven dissektion medium </td></tr><tr><td> Føtalt kalveserum </td><td> Atlanta Biologicals </td><td> S11150 </td><td> Rygmarven dissektion medium </td></tr><tr><td> Vännäs Saks </td><td> Verdens præcisionsinstrumenter </td><td> 501778 </td><td> Rygmarven dissektion </td></tr><tr><td>&nbsp;</td><td></td><td></td><td></td></tr><tr><td> Kollagen forberedelse </td><td></td><td></td><td></td></tr><tr><td> Rotte-hale kollagen type I </td><td> BD Biosciences </td><td> 354249 </td><td> Eksplantation kultur </td></tr><tr><td> 10X PBS (Steril) </td><td></td><td></td><td> Til Neutralize kollagen </td></tr><tr><td> 1N NaOH (Steril) </td><td></td><td></td><td> For at neutralisere kollagen </td></tr><tr><td> Destilleret vand (Steril) </td><td></td><td></td><td> For at neutralisere kollagen </td></tr><tr><td> pH-indikator papirer (6,0-8,1) </td><td> Whatman </td><td> 2629-990 </td><td> For at kontrollere kollagen pH </td></tr><tr><td> 15 ml koniske rør </td><td> Dot Scientific Inc </td><td> 818-PG </td><td></td></tr><tr><td> 500 μl bred mund pipettespidser </td><td> Dot Scientific Inc </td><td> 119-R100 </td><td> Til pipette kollagen </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Eksplantation kultur </td><td></td><td></td><td></td></tr><tr><td> DMEM/F12 medium </td><td> Sigma </td><td> D8437 </td><td> Eksplantation dyrkningsmedium </td></tr><tr><td> ITS en </td><td> Sigma </td><td> I2521 </td><td> Medium supplement </td&gt; </tr><tr><td> Penicillin-Streptomycin </td><td> Invitrogen </td><td> 15140-122 </td><td> Medium supplement </td></tr><tr><td> CNTF (rotte) </td><td> Sigma </td><td> C3835 </td><td> Medium supplement </td></tr><tr><td> NT-3 (human) </td><td> Sigma </td><td> N1905 </td><td> Medium supplement </td></tr><tr><td> Renset mus Wnt5a </td><td> R &amp; D Systems </td><td> 645-WN-010 </td><td></td></tr><tr><td> Affi-gel blå gel perler </td><td> Bio-Rad </td><td> 153-7302 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Immunhistokemi </td><td></td><td></td><td></td></tr><tr><td> Paraformaldehyd </td><td> Sigma </td><td> P6148 </td><td> Fiksering </td></tr><tr><td> PBS </td><td></td><td></td><td> Skylninger </td></tr><tr><td> Triton X-100 </td><td> Sigma </td><td> T9284 </td><td> Blokerende opløsning </td></tr><tr><td> Natriumazid </td><td> Sigma </td><td> S8032 </td><td> Blokerende opløsning </td></tr><tr><td> Kalveserum </td><td> Invitrogen </td><td> 16170-078 </td><td> Blokerende opløsning </td></tr><tr><td> Muse monoklonalt IgG2b anti-β tubulin 1 + II antistof </td><td> Sigma </td><td> T8535 </td><td> Etiketter celle organer og neurites </td></tr><tr><td> Alexa fluor 488 ged anti-mus IgG2 b-antistof </td><td> Invitrogen </td><td> A21141 </td><td> Registrerer β tubulin antistof </td></tr><tr><td> Teflon mikro spatel </td><td> VWR </td><td> 57949-033 </td><td> Frigivelse kollagen gel fra godt </td></tr></tbody></table>

dissection and culture of chick statoacoustic ganglion and spinal cord explants in collagen gels for neurite outgrowth assays

Den sanseorganer af kylling indre øre er innerveres af de perifere processer statoacoustic ganglion (SAG) neuroner. Sanseorgan innervation afhænger af en kombination af axon vejledning tidskoder 1 og overlevelsesfaktorer 2 placeret langs bane voksende axoner og / eller inden for deres sanseorgan mål. For eksempel genereret funktionel interferens med en klassisk Axon vejledning signalvejen, semaphorin-neuropilin, misrouting af eksotiske axoner 3. Også flere vækstfaktorer til udtryk i den sensoriske mål i det indre øre, herunder neurotrophin-3 (NT-3) og Brain Afledte Neurotrope Factor (BDNF), er blevet manipuleret i transgene dyr, igen fører til misrouting af SAG axoner 4. Disse samme molekyler fremme både overlevelse og neurite udvækst af chick SAG neuroner in vitro 5,6. Her vil vi beskrive og demonstrere in vitro-metode vi i øjeblikket usynge for at teste reaktionsevne chick SAG neurites til opløselige proteiner, herunder kendte morphogens som Wnts, samt vækstfaktorer, som er vigtige for at fremme SAG neurite udvækst og neuron overlevelse. Ved hjælp af denne model system, vi håber at kunne drage konklusioner om de virkninger, som udskilles ligander kan udøve på SAG neuron overlevelse og neurite udvækst. SAG eksplanteret er dissekeret i embryonale dag 4 (E4) og kultiveret i tre-dimensionelle kollagen gel under serum-fri betingelser for 24 timer. Først er neurite reaktionsevne testet af dyrkning eksplanteret med protein-suppleret medium. Så, for at spørge, om punktkilder udskilles ligander kan have retningsbestemte effekter på neurite outgrowth, er eksplanteret co-kulturperler med protein-belagt perler og analyseret for evne til perle til lokalt at fremme eller hæmme udvækst. Vi har også omfatter en demonstration af dissektion (modificeret protokol 7) og kultur E6 rygmarven eksplanteret. Virutinemæssigt bruger rygmarven eksplanteret at bekræfte bioaktivitet af proteiner og protein-gennemblødt perler, og at kontrollere arter krydsreaktivitet med chick væv, under den samme kultur betingelser som SAG eksplanteret. Disse in vitro assays er bekvemt for hurtig screening for molekyler, der udøver trofiske (overlevelse) eller Tropic (retningsbestemt) virkninger på SAG neuroner, især før udførelse af undersøgelser in vivo. Desuden er denne metode giver mulighed for afprøvning af enkelte molekyler under serumfrit betingelser med høj neuron overlevelse 8.

Watch this Scientific Journal Video about Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays at JoVE.com

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

Vi viser, hvordan du dissekere og kultur chick E4 statoacoustic ganglion og E6 rygmarven eksplanteret. Eksplanteret dyrkes under serumfrit betingelser i 3D kollagen geler i 24 timer. Neurite lydhørhed er testet med vækstfaktor-suppleret medium og med protein-belagt perler.

Dissektion og Kultur af Chick Statoacoustic Ganglion og rygmarven eksplanteret i Kollagen Gels for neurite udvækst Analyser

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

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