Name: neural explant cultures from xenopus laevis
Uploaded: 2012-10-15T12:00:00
Description: Watch this Scientific Journal Video about Neural Explant Cultures from Xenopus laevis at JoVE.com

Forfatterne ønsker å takke Bob Freeman for trening og Kirschner lab for bruk av frosken anlegget, og medlemmer av Van Vactor lab for støtte. Vi takker Nikon Imaging Center ved Harvard Medical School for å få hjelp med lysmikroskopi for bildene i Figur 1. Dette arbeidet ble finansiert av følgende: NRSA NIH fellesskap og NIH K99 fellesskap å LAL, Basic Science Partnership finansiering ( <a href="https://bsp.med.harvard.edu/" target="_blank">https://bsp.med.harvard.edu/</a> ) til AEF, og NIH RO1 NS035909 til DVV

Merk: Vi beskriver trinnene i de to første avsnittene bare i korthet, som utmerket protokoller med detaljert informasjon er publisert andre steder som fokuserer mer spesifikt på disse trinnene (f.eks 8-12). I tillegg har den generelle protokoll med å jobbe med Xenopus spinal nevroner i levende cellekultur er tidligere publisert i en detaljert metoder artikkel 8. Vi anbefaler å gjennomgå at artikkelen som et supplement til denne videoen, men her vi gir god nok informasjon til ...

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Xenopus laevis nevrale explants sende ut neurites i en svært robust måte med 24 timer etter plating på laminin / poly-lysin substrat hvis forholdene ligger til rette. Med dette substrat, vekst kjegler er svært motile og kan oppnå axon lengder opp til 1 mm, som strekker seg i alle retninger utover fra eksplantasjon, selv om typiske lengder er 100 mikrometer eller mer. Hvis neurites ikke vokse ut, det er et begrenset antall grunner for at dette skal være tilfelle. En mulighet er at de nevrale explants ikke ...

<table border="1"><tbody><tr><td> Navn på Reagens </td><td> Selskapet </td><td> Katalognummer </td></tr><tr><td> Choriongonadotropin </td><td> Argent Chemical Laboratories </td><td> C-HCG-ON-10 </td></tr><tr><td> Cysteine </td><td> Sigma-Aldrich </td><td> 52-90-4 </td></tr><tr><td> mMessage mMachine kit </td><td> Ambion </td><td> AM1340 </td></tr><tr><td> Kapillær Borosil Needles 1,2 mm (OD) x 0,9 mm (ID) </td><td> FHC </td><td> 30-31-0 </td></tr><tr><td> Ficoll </td><td> Sigma </td><td> F2637 </td></tr><tr><td> Dumont # 5 Biologie Inox tang </td><td> Fin Science Verktøy </td><td> 11252-20 </td></tr><tr><td> Kollagenase </td><td> Sigma-Aldrich </td><td> 9001-12-1 </td></tr><tr><td> Mattek retter </td><td> Mat Tek Corporation </td><td> P35G-1.5-14-C </td></tr><tr><td> L-15 </td><td> Invitrogen </td><td> 21083-027 </td></tr><tr><td> Poly-l-lysine </td><td> Sigma </td><td> P-1399 </td></tr><tr><td> Laminin </td><td> Sigma </td><td> L2020 </td></tr><tr><td> NT3 </td><td> Sigma </td><td> N1905 </td></tr><tr><td> BDNF </td><td> Sigma </td><td> B3795 </td></tr></tbody></table>

neural explant cultures from xenopus laevis

Den komplekse prosessen med axon veiledning er stor grad drevet av veksten membran, som er den dynamiske motile strukturen på spissen av den voksende axon. Under axon utvekst, må veksten kjegle integrere flere kilder til veiledning cue informasjon å modulere sin cytoskjelettet for å drive veksten kjegle fremover og nøyaktig navigere for å finne sine spesifikke mål en. Hvordan denne integrasjonen skjer på cytoskeletal nivået fortsatt voksende, og undersøkelse av cytoskeletal protein og effektor dynamikk innenfor veksten kjegle kan tillate klarlegging av disse mekanismene. Xenopus laevis vekst membranene er stor nok (10-30 mikron i diameter) for å utføre høy -oppløsning levende avbildning av cytoskeletal dynamikk (f.eks 2-4) og er lett å isolere og manipulere i en lab miljø i forhold til andre virveldyr. Frosken er en klassisk modell system for utvikling nevrobiologi studier og viktige tidlige innsikt i vekst kjegle microtubule dynamikk ble opprinnelig funnet ved hjelp av dette systemet 5-7. I denne metoden 8, er egg samlet og befruktet in vitro, injisert med RNA som koder for fluorescensmerket tagged cytoskeletal fusjonsproteiner eller andre konstruksjoner for å manipulere genekspresjon, og deretter tillatt å utvikle seg til nevralrøret scenen. Neural rør er isolert ved disseksjon og deretter dyrkes, og vekst kjegler på outgrowing neurites er avbildes. I denne artikkelen beskriver vi hvordan du utfører denne metoden, der målet er å kultur Xenopus laevis vekst kjegler for påfølgende høy oppløsning bildeanalyse. Mens vi gi eksempel på + TIP fusion protein EB1-GFP kan denne metoden brukes til en rekke proteiner for å belyse deres atferd innenfor veksten kjegle.

Watch this Scientific Journal Video about Neural Explant Cultures from Xenopus laevis at JoVE.com

Neural Explant Cultures from Xenopus laevis

Dyrking nevrale explants fra dissekert<em&gt; Xenopus laevis</em&gt; Embryo som uttrykker fluorescerende fusjonsproteiner tillater avbildning av vekst cone cytoskeletal dynamikk.

Neural Eksplantasjon kulturer fra<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

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Neural Explant Cultures from Xenopus laevis

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

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

Isolering og kultur neural crest Celler fra Embryonic 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 ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Disseksjon, kultur, og analyse av<em&gt; Xenopus laevis</em&gt; Embryonic Retinal Tissue

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Samtidig Pre-og postsynaptiske Elektrofysiologisk Opptak fra<em&gt; Xenopus</em&gt; Nerve-muskel Co-kulturer

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

Neural Circuit Recording from an Intact Cockroach Nervous System

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The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

En mus, to kulturer: Isolation and Culture of Adult nevrale stamceller fra de to nevrogene Soner Individuell Mus

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

En enkel Behavioral analysen for Testing visuell funksjon i<em&gt; Xenopus laevis</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Opptak Temperatur-indusert neuronal aktivitet gjennom Overvåking Kalsium endringer i luktelappen av<em&gt; Xenopus laevis</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

Avledning av leptomeninges eksplantering kulturer fra postmortem menneskelige hjerne givere

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of ...