Name: isolation and culture of rat embryonic neural cells: a quick protocol
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Wij danken Jonna Ellis voor de redactionele ondersteuning. Het beschreven project werd ondersteund door Award Aantal R01MH079751 (PI: F. Peruzzi) van het National Institute of Mental Health. De inhoud is uitsluitend de verantwoordelijkheid van de auteurs en niet noodzakelijkerwijs het officiële standpunt van het National Institute of Mental Health en de National Institutes of Health.

1. Poly-D-Lysine (PDL): Voorbereiding <ol><li> Voeg 5 ml steriele DDH 2 O tot 5 mg van het PDL om een voorraad oplossing van 1 mg / ml te verkrijgen. </li><li> Meng voorraadoplossing door pipetteren meerdere malen. </li><li> Onmiddellijk gebruiken of op te slaan Poly-D-Lysine oplossing bij 2-8 ° C. </li></ol> 2. Poly-D-Lysine (PDL): Coating Plastic Cel Cultuur Dishes <ol><li> Verdun de PDL voorraadoplossing met steriel DDH 2 O de uiteindelijke concentratie van 10 ug / m...

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	<li>Aprea, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubulin-mediated+binding+of+human+immunodeficiency+virus-1+Tat+to+the+cytoskeleton+causes+proteasomal-dependent+degradation+of+microtubule-associated+protein+2+and+neuronal+damage.">Tubulin-mediated binding of human immunodeficiency virus-1 Tat to the cytoskeleton causes proteasomal-dependent degradation of microtubule-associated protein 2 and neuronal damage.</a> J. Neurosci. 26, 4054-4062 (2006).</li><li>Kivell, B. M., McDonald, F. J., Miller, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+culture+of+rat+post-natal+and+fetal+brainstem+neurons.">Serum-free culture of rat post-natal and fetal brainstem neurons.</a> Brain Res. Dev. Brain Res. 120, 199-210 (2000).</li><li>Kivell, B. M., McDonald, F. J., Miller, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+serum-free+culture+of+late+fetal+and+early+postnatal+rat+brainstem+neurons.">Method for serum-free culture of late fetal and early postnatal rat brainstem neurons.</a> Brain Res. Brain Res. Protoc. 6, 91-99 (2001).</li><li>Brewer, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+B27/neurobasal+medium+supports+differentiated+growth+of+neurons+from+the+striatum,+substantia+nigra,+septum,+cerebral+cortex,+cerebellum,+and+dentate+gyrus.">Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus.</a> J. Neurosci. Res. 42, 674-683 (1995).</li><li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing nerve cells. , (1998).</li><li>Eletto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+SNAP25+expression+by+HIV-1+Tat+involves+the+activity+of+mir-128a.">Inhibition of SNAP25 expression by HIV-1 Tat involves the activity of mir-128a.</a> J. Cell Physiol. 216, 764-770 (2008).</li><li>Gualco, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGF-IR-dependent+expression+of+Survivin+is+required+for+T-antigen-mediated+protection+from+apoptosis+and+proliferation+of+neural+progenitors.">IGF-IR-dependent expression of Survivin is required for T-antigen-mediated protection from apoptosis and proliferation of neural progenitors.</a> Cell Death Differ. 17, 439-451 (2010).</li><li>Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+differentiation+of+adult+neuronal+progenitor+cells+transplanted+to+the+adult+brain.">Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain.</a> Proc. Natl. Acad. Sci. U.S.A. 92, 11879-11883 (1995).</li><li>Keyser, D. O., Pellmar, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+transmission+in+the+hippocampus:+critical+role+for+glial+cells.">Synaptic transmission in the hippocampus: critical role for glial cells.</a> Glia. 10, 237-243 (1994).</li><li>Pfrieger, F. W., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+efficacy+enhanced+by+glial+cells+in+vitro.">Synaptic efficacy enhanced by glial cells in vitro.</a> Science. 277, 1684-1687 (1997).</li></ol>

De werkwijze van ontleding en cultuur rat hippocampale en corticale neuronen beschreven kan uitvoeren experimenten met nagenoeg zuiver neuronale kweken gekweekt in een chemisch gedefinieerd medium (figuur 3). Hoewel protocollen voor het kweken van nagenoeg zuivere neuronen in serum-vrij medium zijn eerder beschreven 2,3,4 er belangrijke wijzigingen in onze methode. Anders dan de traditionele protocollen (dat wil zeggen Banker et al..) 5, hebben we vervangen door trypsine met TrypLE...

<table border="1"><tbody><tr><td> Reagens </td><td> Concentratie </td></tr><tr><td> Neurobasal </td><td> 98% </td></tr><tr><td> B27 </td><td> 2% </td></tr><tr><td> Glutamax </td><td> 0,5 mM </td></tr></tbody></table> Tabel I. Neurobasal/B27 volledig medium. <table border="1"><tbody><tr><td> Reagens </td><td> Concentratie </td></tr><tr><td> Glucose </td><td> 16 mM </td></tr><tr><td> Sucrose </td><td> 22 mM </td></tr><tr><td> HEPES </td><td> 10 mM </td></tr><tr><td> NaCl </td><td> 160 mM </td></tr><tr><td> KCl </td><td> 5 mM </td></tr><tr><td> Na 2 HPO 4 </td><td> 1 mM </td></tr><tr><td> KH 2 PO 2 </td><td> 0,22 mM </td></tr><tr><td> Gentamicine </td><td> 50 ug / ml </td></tr&gt; <tr><td> Fungizone </td><td> 250 ng / ml </td></tr><tr><td> pH </td><td> 7,4 </td></tr><tr><td> Osmolariteit </td><td> 320-330 mOsm </td></tr></tbody></table> Tabel II. Dissection medium. <table border="1"><tbody><tr><td> Reagens </td><td> Volume (il) </td></tr><tr><td> Neurobasal/B27 complete medium </td><td> 240 </td></tr><tr><td> Trypanblauw Stain 0,4% </td><td> 250 </td></tr><tr><td> Totaal </td><td> 490 </td></tr></tbody></table> Tabel III. 50x tellen oplossing. <table border="1"><tbody><tr><td> Reagens </td><td> Vennootschap </td><td> Cat. aantal </td></tr><tr><td> Hibernate E </td><td> Brainbits </td><td> 767171 </td></tr><tr><td> Neurobasal </td><td> Gibco, Invitrogen </td><td> 21103-049 </td></tr><tr><td> B27 </td><td> Gibco, Invitrogen </td><td> 17504-044 </td></tr><tr><td> Fungizone </td><td> Gibco, Invitrogen </td><td> 15290-018 </td></tr><tr><td> Gentamicinesulfaat </td><td> Sigma Aldrich </td><td> G1264 </td></tr><tr><td> Glutamax 200 mM </td><td> Gibco, Invitrogen </td><td> 35050 </td></tr><tr><td> TrypLE Express w / o fenolrood </td><td> Gibco, Invitrogen </td><td> 12604 </td></tr><tr><td> Cytosine-β-D-arabinofuranoside hydrochloride </td><td> Sigma Aldrich </td><td> C6645 </td></tr><tr><td> Poly-D-lysine </td><td> Sigma Aldrich </td><td> P6407 </td></tr><tr><td> Laminine 1 mg / ml </td><td> Millipore </td><td> CC095 </td></tr><tr><td> HEPES </td><td> Sigma Aldrich </td><td> H3375 </td></tr><tr><td> Trypanblauw Stain 0,4% </td><td> Gibco, Invitrogen </td><td> 15250 </td></tr></tbody></table&gt; Tabel IV. Specifieke reagentia. <table border="1"><tbody><tr><td> Uitrusting </td><td> Vennootschap </td><td> Cat. aantal </td></tr><tr><td> Stereo Microscoop </td><td> Olympus </td><td> SZ61 </td></tr><tr><td> Grote tang </td><td> FST </td><td> 11022-14 </td></tr><tr><td> Fijne punt tang </td><td> Moria </td><td> MC40B </td></tr><tr><td> Micro fijne punt tang </td><td> Moria </td><td> MC31 </td></tr><tr><td> Haarscherpe schaar </td><td> Roboz </td><td> RS-6820 </td></tr><tr><td> Micro ontleden schaar </td><td> FST </td><td> 91460-11 </td></tr><tr><td> Micro ontleden Gebogen schaar </td><td> FST </td><td> 14067-11 </td></tr><tr><td> Glas 2-kamer dia&#39;s </td><td> Lab-Tek </td><td> 154461 </td></tr><tr><td> 60 mm schaaltjes </td&gt; <td> BD Falcon </td><td> 353002 </td></tr><tr><td> 100 mm schaaltjes </td><td> Corning </td><td> 430167 </td></tr><tr><td> 15 ml buizen </td><td> BD Falcon </td><td> 352099 </td></tr><tr><td> 1,5 ml cryo-tube flacon </td><td> Nunc </td><td> 375353 </td></tr></tbody></table> Tabel V. specifieke apparatuur.

isolation and culture of rat embryonic neural cells: a quick protocol

We zijn het beschrijven van een snelle methode om zich te distantiëren en cultuur hippocampus of corticale neuronen van E15-17 rat embryo&#39;s. De procedure kan met succes worden toegepast voor de isolatie van muizen en menselijke primaire neuronen en neurale progenitoren. Los neuronen worden bijgehouden in serum-vrij medium tot enkele weken. Deze culturen gebruikt worden voor nucleofectie, immunocytochemie, nucleïnezuren bereiding en elektrofysiologie. Oudere neuronale kweken ook worden getransfecteerd met een goede rendement van lentivirale transductie en minder efficiënt, met calciumfosfaat of lipide-gebaseerde methoden zoals lipofectamine.

Watch this Scientific Journal Video about Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol at JoVE.com

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We beschrijven een snelle methode voor het isoleren en cultuur hippocampus en corticale neuronen van knaagdieren embryo&#39;s. Dit protocol stelt ons in staat uit te voeren experimenten waarbij bijna zuivere neuronale culturen zijn vereist.

Isolatie en Cultuur van Rat Embryonale neurale cellen: een Quick-protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

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