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 arbeidet ble finansiert av National Institutes of Health Grant RO1DC002756 og Purdue Research Foundation. Vi takker Doris Wu og Wiese Chang for råd med eksperimenter og Rodney McPhail for hjelp med figurer.

Oppskrifter 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> Vann (Tissue kultur grade) </td></tr></tbody></table> Merk: Juster pH til 7,4. Ta endelig volum til 1000 ml med vann. 10X PBS <table border...

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Vi presenterer en metode for å dissekere og kultur E4 SAG og E6 ryggmargen explants, fra kylling, under serum-free forhold. Denne prosedyren brukes i dag i vårt laboratorium for å studere effekter av ulike utskilles ligander på SAG neuron overlevelse og neurite utvekst. Novel aspekter av denne protokollen inkluderer bruk av et serum-free system for dyrking explants og bruk av perler dynket i vekstfaktorer for å studere effekter på SAG neurite utvekst. En perle metode som oss har blitt beskrevet for dama ryggmargen...

<table border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Katalognummer </td><td> Kommentar </td></tr><tr><td> Utstyr </td><td></td><td></td><td></td></tr><tr><td> Disseksjon mikroskop </td><td></td><td></td><td> Vi bruker en Leica M80 stereomikroskop med lysfelt basen belysning </td></tr><tr><td> Slide varmere </td><td> CS &amp; E. </td><td></td><td> Kollagen polymerisasjon </td></tr><tr><td> Chicken egg inkubator </td><td></td><td></td><td></td></tr><tr><td> 37 ° C / 5% CO 2 fuktet cellekultur inkubator </td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Disseksjon 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 plate </td><td> Corning </td><td> 3526 </td><td></td></tr><tr><td> # 5 Dumont tang </td><td> Fin Science Verktøy </td><td> 11252-30 </td><td></td></tr><tr><td> # 55 Dumont tang </td><td> Fin Science Verktøy </td><td> 11295-51 </td><td></td></tr><tr><td> Rustfritt stål dissekerende pins </td><td> Fin Science Verktøy </td><td> 26002-10 </td><td> Embryo låsing, fine disseksjon </td></tr><tr><td> Moria perforert skje </td><td> Fin Science Verktøy </td><td> 10370-17 </td><td> Embryo høste / overføring </td></tr><tr><td> 200 mL bred munn pipettér tips </td><td> Dot Scientific Inc </td><td> 118-96R </td><td> Eksplantering transfer </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> E4 og E6 kylling egg </td><td></td><td></td><td> &amp; Nbsp; </td></tr><tr><td> Chick Ringer-løsning </td><td></td><td></td><td> Embryo høste </td></tr><tr><td> HBSS </td><td> Sigma </td><td> H8264 </td><td> SAG disseksjon </td></tr><tr><td> L15 medium </td><td> Sigma </td><td> L5520 </td><td> Ryggmargen disseksjon medium </td></tr><tr><td> Kalvefoster serum </td><td> Atlanta Biologicals </td><td> S11150 </td><td> Ryggmargen disseksjon medium </td></tr><tr><td> Vännäs Saks </td><td> Verden presisjonsinstrumenter </td><td> 501778 </td><td> Ryggmargen disseksjon </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> Rat-tail kollagen Type I </td><td> BD Biosciences </td><td> 354249 </td><td> Eksplantering kultur </td></tr><tr><td> 10X PBS (sterilt) </td><td></td><td></td><td> Til neutralize kollagen </td></tr><tr><td> 1N NaOH (sterilt) </td><td></td><td></td><td> Å nøytralisere kollagen </td></tr><tr><td> Destillert vann (sterilt) </td><td></td><td></td><td> Å nøytralisere kollagen </td></tr><tr><td> pH indikator papers (6,0 til 8,1) </td><td> Whatman </td><td> 2629-990 </td><td> For å sjekke kollagen pH </td></tr><tr><td> 15 ml konisk rør </td><td> Dot Scientific Inc </td><td> 818-PG </td><td></td></tr><tr><td> 500 mL bred munn pipettér tips </td><td> Dot Scientific Inc </td><td> 119-R100 </td><td> Til pipettér kollagen </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Eksplantering kultur </td><td></td><td></td><td></td></tr><tr><td> DMEM/F12 medium </td><td> Sigma </td><td> D8437 </td><td> Eksplantering kultur medium </td></tr><tr><td> SITT 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> Immunhistokjemi </td><td></td><td></td><td></td></tr><tr><td> Paraformaldehyde </td><td> Sigma </td><td> P6148 </td><td> Fiksering </td></tr><tr><td> PBS </td><td></td><td></td><td> Skyllinger </td></tr><tr><td> Triton X-100 </td><td> Sigma </td><td> T9284 </td><td> Blokkering løsning </td></tr><tr><td> Sodiumazid </td><td> Sigma </td><td> S8032 </td><td> Blokkering løsning </td></tr><tr><td> Calf serum </td><td> Invitrogen </td><td> 16170-078 </td><td> Blokkering løsning </td></tr><tr><td> Monoklonalt IgG2b anti-β tubulin 1 + II antistoff </td><td> Sigma </td><td> T8535 </td><td> Etiketter celle organer og neurites </td></tr><tr><td> Alexa Fluor 488 geit anti-mus IgG2 b antistoff </td><td> Invitrogen </td><td> A21141 </td><td> Oppdager β tubulin antistoff </td></tr><tr><td> Teflon micro slikkepott </td><td> VWR </td><td> 57949-033 </td><td> Slipp kollagen gels fra brønn </td></tr></tbody></table>

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

Den sensoriske organer kylling indre øret er innerverte av perifere prosesser statoacoustic ganglion (SAG) nerveceller. Sensoriske organ innervasjon er avhengig av en kombinasjon av axon veiledning pekepinner 1 og overlevelse faktorer 2 ligger langs banen voksende axoner og / eller innenfor de sensoriske organ mål. For eksempel generert funksjonelle forstyrrelser med en klassisk axon veiledning signalveien, semaphorin-neuropilin, misrouting av otic axons 3. Også flere vekstfaktorer uttrykt i sensoriske mål i det indre øret, inkludert Neurotrophin-3 (NT-3) og Brain Avledet neurotrophic Factor (BDNF), har blitt manipulert i transgene dyr, igjen fører til misrouting av SAG axons fire. De samme molekylene fremme både overlevelse og neurite utvekst av chick SAG nerveceller in vitro 5,6. Her beskriver vi og demonstrere in vitro metode er vi for tiden usynge for å teste responsen til chick SAG neurites til løselige proteiner, inkludert kjente morphogens som Wnts, samt vekstfaktorer som er viktige for å fremme SAG neurite utvekst og neuron overlevelse. Ved hjelp av denne modellen system, håper vi å trekke konklusjoner om effekter som utskilles ligander kan øve på SAG neuron overlevelse og neurite utvekst. SAG explants er dissekert på embryonale dag 4 (E4) og kultivert i tredimensjonale kollagen gel henhold til serum-frie forhold i 24 timer. Først er neurite respons testet av dyrkning explants med protein-supplert medium. Så, for å spørre om punktkilder av utskilles ligander kan ha retningsbestemt effekter på neurite utvekst, er explants co-kultiverte med protein-belagt perler og analysert for evne til perle til lokalt fremme eller hemme utvekst. Vi inkluderer også en demonstrasjon av disseksjon (modifisert protokoll 7) og kultur av E6 ryggmargen explants. Virutinemessig bruk ryggmargen explants å bekrefte bioaktivitet av proteiner og protein-gjennomvåt perler, og for å verifisere arter kryssreaksjon med kylling vev, under samme kultur vilkår som SAG explants. Disse in vitro er praktisk for raskt screening for molekyler som utøver trofisk (overlevelse) eller tropic (retningsbestemt) effekter på SAG nervecellene, spesielt før du utfører studier in vivo. Videre tillater denne metoden testing av enkelte molekyler i henhold til serum-frie forhold, med høy 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 å dissekere og kultur chick E4 statoacoustic ganglion og E6 ryggmargen explants. Explants dyrkes i henhold til serum-frie forhold i 3D kollagen gels i 24 timer. Neurite respons er testet med vekstfaktor-supplert medium og med protein-belagt perler.

Disseksjon og kultur av Chick Statoacoustic ganglion og Spinal Cord explants i Collagen Gels for neurite utvekst Assays

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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