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 vil gerne takke Bob Freeman for uddannelse og Kirschner laboratorium for brug af frøen rum og medlemmer af Van Vactor lab for support. Vi takker Nikon Imaging Center ved Harvard Medical School for assistance med lysmikroskopi for billederne i figur 1. Dette arbejde blev finansieret af følgende: NRSA NIH fællesskab og NIH K99 fællesskab til 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

Bemærk: Vi beskriver trinene i de to første afsnit kun i kort, som fremragende protokoller med detaljerede oplysninger er blevet offentliggjort andetsteds, der fokuserer mere specifikt på disse trin (f.eks 8-12). Derudover har den generelle protokol at arbejde med Xenopus spinale neuroner i levende cellekultur tidligere blevet offentliggjort i et detaljeret metoder artikel 8. Vi stærkt anbefale, at artiklen som et supplement til denne video, men her vi giver nok information ti...

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Xenopus laevis neurale eksplantater sende neuritter i en meget robust måde ved 24 timer efter udpladning på laminin / poly-lysin substrat, hvis forholdene er passende. Med dette substrat, er vækstkegler meget motile og kan opnå axon længder på op til 1 mm, der strækker sig i alle retninger udad fra eksplantatet, men typiske længder er 100 um eller mere. Hvis neuritter ikke vokser ud, er der et begrænset antal grunde til, at dette er tilfældet. En mulighed er, at de neurale eksplantater ikke korrekt ho...

<table border="1"><tbody><tr><td> Navn reagens </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> Chorion gonadotropin </td><td> Argent Chemical Laboratories </td><td> C-HCG-ON-10 </td></tr><tr><td> Cystein </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 Tools </td><td> 11.252-20 </td></tr><tr><td> Collagenase </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 komplicerede proces med axon vejledning er i høj grad drevet af væksten kegle, hvilket er den dynamiske motile struktur på spidsen af ​​den voksende axon. Under Axon udvækst må væksten kegle integrere flere kilder til vejledning cue information at modulere sin cytoskeleton for at drive væksten kegle fremad og præcist navigere for at finde de specifikke mål 1. Hvordan denne integration forekommer ved cytoskeletal niveau stadig opstår, og undersøgelse af cytoskeletprotein og effektor dynamik i vækst kegle kan tillade belysning af disse mekanismer. Xenopus laevis vækstkegler er stor nok (10 til 30 mikron i diameter) for at udføre høj opløsning direkte billedvisning af cytoskeletale dynamik (fx 2-4) og er lette at isolere og manipulere i et laboratorium indstilling i forhold til andre hvirveldyr. Frøen er en klassisk model for udviklingsmæssige neurobiologi undersøgelser, og vigtige tidlige indsigt i vækst kegle microtubule dynamik blev oprindeligt fundet ved hjælp af dette system 5-7. I denne metode 8 bliver æg opsamles og befrugtes in vitro, injiceret med RNA, der koder fluorescerende mærkede cytoskeletale fusionsproteiner eller andre konstruktioner til at manipulere genekspressionen, og fik derefter lov til at udvikle sig til neuralrøret fase. Neurale rør isoleres ved dissektion og derefter dyrkes, og vækstkegler på outgrowing neuritter afbildes. I denne artikel beskriver vi, hvordan at udføre denne fremgangsmåde, hvis formål er at kultur Xenopus laevis vækstkegler til efterfølgende høj opløsning billedanalyse. Mens vi giver som eksempel + TIP fusionsprotein EB1-GFP, kan denne fremgangsmåde anvendes til ethvert antal proteiner at belyse deres adfærd i vækst kegle.

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

Neural Explant Cultures from Xenopus laevis

Dyrkning af neurale eksplantater fra dissekeret<em&gt; Xenopus laevis</em&gt; Embryoner, der udtrykker fluorescerende fusionsproteiner tillader afbildning af vækst kegle cytoskeletale dynamik.

Neurale eksplantatkulturer 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 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 ...

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

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

Dissektion, Kultur, og analyse af<em&gt; Xenopus laevis</em&gt; Embryonic retinavæv

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 Post-synaptiske Elektrofysiologiske Optagelse 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, ...

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Neural Circuit Optagelse fra en intakt Kakerlak Nervous System

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

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En mus, to kulturer: isolering og dyrkning af voksne neurale stamceller fra Two Neurogene zoner Individuel 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 simpel Behavioral Assay for Testing Visual Funktion 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

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Optagelse Temperatur-induceret neuronal aktivitet gennem Overvågning Calcium Ændringer i det olfaktoriske pære af<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

Udledning af leptomeninges eksplantatkulturer fra Postmortem menneskehjerne donorer

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