Name: isolation and culture of rat embryonic neural cells: a quick protocol
Uploaded: 2012-05-24T12:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol at JoVE.com

Agradecemos Jonna Ellis para a assistência editorial. O projeto descrito foi apoiada por número Prêmio R01MH079751 (PI: F. Peruzzi) do Instituto Nacional de Saúde Mental. O conteúdo é da exclusiva responsabilidade dos autores e não representam necessariamente as opiniões oficiais do Instituto Nacional de Saúde Mental ou o National Institutes of Health.

1. Poli-D-lisina (PDL): Preparação <ol><li> Adicionar 5 ml de ddH2O estéril a 5 mg de PDL para se obter uma solução stock de 1 mg / ml. </li><li> Misture solução estoque pipetando várias vezes. </li><li> Use imediatamente ou guarde Poly-D-lisina solução a 2-8 ° C. </li></ol> 2. Poli-D-lisina (PDL): Revestimento de plástico pratos de cultura de células <ol><li> Dilui-se a solução de reserva com PDL estéril ddH2O para a concentração final de 10 ug / ml. <...

<ol>
	<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>

O método de dissecção e cultura de neurónios do hipocampo de ratos e cortical aqui descrito permite a realização de experiências usando quase puros culturas neuronais cultivadas num meio quimicamente definido (Figura 3). Embora os protocolos para os neurónios de cultura quase puros em meios isentos de soro foram anteriormente descritos 2,3,4, há alterações importantes feitas em nosso método. Diferente de protocolos tradicionais (isto é, Banker et al.) 5, temos substitu...

<table border="1"><tbody><tr><td> Reagente </td><td> Concentração </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> Quadro I. Neurobasal/B27 meio completo. <table border="1"><tbody><tr><td> Reagente </td><td> Concentração </td></tr><tr><td> Glicose </td><td> 16 mM </td></tr><tr><td> Sacarose </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> Gentamicina </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> Osmolaridade </td><td> 320-330 mOsm </td></tr></tbody></table> Tabela II. Meio de dissecção. <table border="1"><tbody><tr><td> Reagente </td><td> Volume (uL) </td></tr><tr><td> Neurobasal/B27 meio completo </td><td> 240 </td></tr><tr><td> Trypan azul 0,4% </td><td> 250 </td></tr><tr><td> Total </td><td> 490 </td></tr></tbody></table> Tabela III. 50x Contando solução. <table border="1"><tbody><tr><td> Reagente </td><td> Companhia </td><td> Cat. número </td></tr><tr><td> E Hibernate </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> De sulfato de gentamicina </td><td> Sigma Aldrich </td><td> G1264 </td></tr><tr><td> Glutamax mM 200 </td><td> Gibco, Invitrogen </td><td> 35050 </td></tr><tr><td> TrypLE expresso vermelho w / o fenol </td><td> Gibco, Invitrogen </td><td> 12604 </td></tr><tr><td> Citosina-β-D-arabinofuranoside cloridrato </td><td> Sigma Aldrich </td><td> C6645 </td></tr><tr><td> Poli-D-Lisina </td><td> Sigma Aldrich </td><td> P6407 </td></tr><tr><td> A laminina 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> Trypan azul 0,4% </td><td> Gibco, Invitrogen </td><td> 15250 </td></tr></tbody></table&gt; Tabela IV. Reagentes específicos. <table border="1"><tbody><tr><td> Equipamento </td><td> Companhia </td><td> Cat. número </td></tr><tr><td> Microscópio estéreo </td><td> Olimpo </td><td> SZ61 </td></tr><tr><td> Pinças grandes </td><td> FST </td><td> 11022-14 </td></tr><tr><td> Pinça de ponta fina </td><td> Moria </td><td> MC40B </td></tr><tr><td> Micro pinça de ponta fina </td><td> Moria </td><td> MC31 </td></tr><tr><td> Afiadas tesouras </td><td> Roboz </td><td> RS-6820 </td></tr><tr><td> Micro tesoura de dissecção </td><td> FST </td><td> 91460-11 </td></tr><tr><td> Dissecando Micro tesoura curva </td><td> FST </td><td> 14067-11 </td></tr><tr><td> Vidro 2-câmara lâminas </td><td> Lab-Tek </td><td> 154461 </td></tr><tr><td> 60 pratos mm </td&gt; <td> BD Falcon </td><td> 353002 </td></tr><tr><td> Placas de 100 mm </td><td> Corning </td><td> 430167 </td></tr><tr><td> 15 ml tubos </td><td> BD Falcon </td><td> 352099 </td></tr><tr><td> 1,5 ml frasco crio-tubo </td><td> Nunc </td><td> 375353 </td></tr></tbody></table> Equipamentos Quadro V. específico.

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

Estamos descrevendo um método rápido para dissociar cultura e os neurônios do hipocampo ou cortical a partir de embriões de ratos E15-17. O procedimento pode ser aplicado com sucesso para o isolamento de rato e humanos neurónios primários e progenitores neurais. Neurónios dissociadas são mantidas em meio isento de soro até várias semanas. Estas culturas podem ser utilizados para nucleofection, imunocitoquímica, a preparação de ácidos nucleicos, bem como electrofisiologia. Mais velhos culturas neuronais também podem ser transfectadas com uma taxa de uma boa eficiência de transdução por lentivirus e, de forma menos eficiente, com fosfato de cálcio ou de lípidos métodos baseados em tais como lipofectamina.

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

Nós descrevemos uma metodologia rápida para isolar e cultivar os neurônios do hipocampo e cortical a partir de embriões de roedores. Este protocolo permite a realização de experimentos em que quase puras culturas neuronais são necessários.

Isolamento e cultivo de células neurais embrionárias de ratos: um protocolo rápido

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

Research

JoVE Journal

Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Dissecção e Cultura de Neurônios da Medula Espinhal comissural embrionárias

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Neurociência

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolamento e Expansão do Rato Adulto células-tronco neurais utilizando o ensaio Neurosphere

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

Estabelecer embrionárias de rato Cultura de Células-tronco neurais utilizando o ensaio Neurosphere

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

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

Neurais Colony-Forming Assay Cell: um ensaio para Discriminar Bona Fide células-tronco neurais de células progenitoras neurais

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). ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Isolamento e Cultura de células da crista neural-tronco de folículos capilares humanos

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Lectina baseado em isolamento e cultura de motoneurônios embrionárias de rato

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

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

Isolamento e cultivo de células da crista neural do tubo neural embrionário murino

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

Dissecção, Cultura, Análise e de<em&gt; Xenopus laevis</em&gt; Tecido embrionário da retina

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 ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Isolamento e cultura de células endoteliais da Embryonic posencéfalo

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

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

Um rato, duas culturas: isolamento e cultivo de células-tronco neurais Adulto de as duas zonas neurogênica Individual Ratos

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