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

Vi tackar Jonna Ellis för redaktionellt hjälp. Projektet beskrivs stöds av Award Antal R01MH079751 (PI: F. Peruzzi) från National Institute of Mental Health. Innehållet är endast ansvar författarnas egna och inte nödvändigtvis officiella ståndpunkter National Institute of Mental Health och National Institutes of Health.

1. Poly-D-lysin (PDL): Framställning <ol><li> Tillsätt 5 ml steril ddHaO 2 O till 5 mg av PDL att erhålla en förrådslösning av 1 mg / ml. </li><li> Blanda stamlösning genom att pipettera flera gånger. </li><li> Använd omedelbart eller förvara Poly-D-Lysin lösning vid 2-8 ° C. </li></ol> 2. Poly-D-lysin (PDL): Beläggning av plast cellodlingsskålar <ol><li> Späd PDL stamlösning med sterilt ddHaO 2 O för att den slutliga koncentrationen av 10 ^ g / ml. </li...

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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>

Metoden enligt dissektion och odling av rått-hippocampus och kortikala neuroner som beskrivs här medger att utföra experiment med användning nästan rena neuronala kulturer som odlats i ett kemiskt definierat medium (figur 3). Även protokoll för odling av nästan rena nervceller i serum-fria medier har tidigare beskrivits 2,3,4, det finns viktiga ändringar som görs i vår metod. Skiljer sig från traditionella protokoll (dvs. Banker et al.) 5 har vi ersatt trypsin med TrypL...

<table border="1"><tbody><tr><td> Reagens </td><td> Koncentration </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> Tabell I. Neurobasal/B27 komplett medium. <table border="1"><tbody><tr><td> Reagens </td><td> Koncentration </td></tr><tr><td> Glukos </td><td> 16 mM </td></tr><tr><td> Sackaros </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> Gentamicin </td><td> 50 | ig / ml </td></tr&gt; <tr><td> Fungizon </td><td> 250 ng / ml </td></tr><tr><td> pH </td><td> 7,4 </td></tr><tr><td> Osmolaritet </td><td> 320-330 mOsm </td></tr></tbody></table> Tabell II. Dissektion mediet. <table border="1"><tbody><tr><td> Reagens </td><td> Volym (^ il) </td></tr><tr><td> Neurobasal/B27 komplett medium </td><td> 240 </td></tr><tr><td> Trypanblått Stain 0,4% </td><td> 250 </td></tr><tr><td> Totalt </td><td> 490 </td></tr></tbody></table> Tabell III. 50x Räkna lösning. <table border="1"><tbody><tr><td> Reagens </td><td> Företaget </td><td> Kat. antal </td></tr><tr><td> Hibernate E </td><td> Brainbits </td><td> 767.171 </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> Fungizon </td><td> Gibco, Invitrogen </td><td> 15290-018 </td></tr><tr><td> Gentamicinsulfat </td><td> Sigma Aldrich </td><td> G1264 </td></tr><tr><td> Glutamax 200 mM </td><td> Gibco, Invitrogen </td><td> 35.050 </td></tr><tr><td> TrypLE Express v / o fenolröd </td><td> Gibco, Invitrogen </td><td> 12.604 </td></tr><tr><td> Cytosin-β-D-arabinofuranosid-hydroklorid </td><td> Sigma Aldrich </td><td> C6645 </td></tr><tr><td> Poly-D-Lysin </td><td> Sigma Aldrich </td><td> P6407 </td></tr><tr><td> Laminin 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> Trypanblått Stain 0,4% </td><td> Gibco, Invitrogen </td><td> 15.250 </td></tr></tbody></table&gt; Tabell IV. Specifika reagens. <table border="1"><tbody><tr><td> Utrustning </td><td> Företaget </td><td> Kat. antal </td></tr><tr><td> Stereo mikroskop </td><td> Olympus </td><td> SZ61 </td></tr><tr><td> Stora Tång </td><td> FST </td><td> 11.022-14 </td></tr><tr><td> Spetsig tång </td><td> Moria </td><td> MC40B </td></tr><tr><td> Micro spetsig tång </td><td> Moria </td><td> MC31 </td></tr><tr><td> Knivskarpa sax </td><td> Roboz </td><td> RS-6820 </td></tr><tr><td> Micro dissekera sax </td><td> FST </td><td> 91.460-11 </td></tr><tr><td> Micro Analysera böjd sax </td><td> FST </td><td> 14.067-11 </td></tr><tr><td> Glas 2-kammare bilder </td><td> Lab-Tek </td><td> 154.461 </td></tr><tr><td> 60 mm skålar </td&gt; <td> BD Falcon </td><td> 353.002 </td></tr><tr><td> 100 mm skålar </td><td> Corning </td><td> 430.167 </td></tr><tr><td> 15 ml rör </td><td> BD Falcon </td><td> 352.099 </td></tr><tr><td> 1,5 ml kryo-röret flaskan </td><td> Nunc </td><td> 375.353 </td></tr></tbody></table> Tabell V. särskild utrustning.

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

Vi beskriver en snabb metod att separera och kultur hippocampus eller kortikal neuroner från E15-17 råttembryon. Förfarandet kan tillämpas med framgång för isolering av mus-och humana primära neuroner och neurala progenitorceller. Dissocierade neuroner bibehålls i serumfritt medium upp till flera veckor. Dessa kulturer kan användas för nucleofection, immunocytokemi, nuklein-syror beredning, liksom elektrofysiologi. Äldre neuronala kulturer kan också transfekteras med en god verkningsgrad genom att lentivirala transduktion och, mindre effektivt, med kalciumfosfat eller lipid-baserade metoder, såsom lipofektamin.

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

Vi beskriver en snabb metod för att isolera och kultur hippocampus och kortikala neuroner från gnagare embryon. Detta protokoll ger oss möjlighet att utföra experiment i vilka nästan rena neuronala kulturer krävs.

Isolering och kultur råttembryonala neurala celler: En snabb protokoll

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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