Name: isolation and culture of hippocampal neurons from prenatal mice
Uploaded: 2012-07-26T12:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Hippocampal Neurons from Prenatal Mice at JoVE.com

Vi takker Dr. Michael Wooten for hans hjælp i udarbejdelsen af ​​manuskriptet. Dette arbejde blev støttet af NIH 2RO1NS033661 (MWW).

1. Set-up før høst <ol><li> For at generere prænatal hvalpe til neuron høst, planlægge parring mellem voksne mus 19 dage før dagen for neuron isolation. (C57BL / 6 mus aldre 2-8 måneder blev brugt i parringer med henblik på at udvikle denne protokol). Vellykket parring kan bekræftes ved påvisning af en vaginal prop i den kvindelige, hjertebanken eller visuel bekræftelse af graviditet. </li><li> Dagen før neuron isolering: <ol class="ualpha"><li> Til immunofluorescens anvendelser coat dækglas i en...

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	<li>Banker, G. A., Cowan, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+hippocampal+neurons+in+dispersed+cell+culture.">Rat hippocampal neurons in dispersed cell culture.</a> Brain Res. 126, 397-442 (1977).</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>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=Isolation+and+culture+of+adult+rat+hippocampal+neurons.">Isolation and culture of adult rat hippocampal neurons.</a> J. Neurosci. Methods. 71, 143-155 (1997).</li><li>Brewer, G. J., Torricelli, J. R., Evege, E. K., Price, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+survival+of+hippocampal+neurons+in+B27-supplemented+Neurobasal,+a+new+serum-free+medium+combination.">Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination.</a> J. Neurosci. Res. 35, 567-576 (1993).</li><li>Burwell, R. D., Saddoris, M. P., Bucci, D. J., Wiig, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corticohippocampal+contributions+to+spatial+and+contextual+learning.">Corticohippocampal contributions to spatial and contextual learning.</a> J. Neurosci. 24, 3826-3836 (2004).</li><li>Gluck, M. A., Myers, C., Meeter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortico-hippocampal+interaction+and+adaptive+stimulus+representation:+a+neurocomputational+theory+of+associative+learning+and+memory.">Cortico-hippocampal interaction and adaptive stimulus representation: a neurocomputational theory of associative learning and memory.</a> Neural Netw. 18, 1265-1279 (2005).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nat. Protoc. 1, 2406-2415 (2006).</li><li>Mao, L., Wang, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gliogenesis+in+the+striatum+of+the+adult+rat:+alteration+in+neural+progenitor+population+after+psychostimulant+exposure.">Gliogenesis in the striatum of the adult rat: alteration in neural progenitor population after psychostimulant exposure.</a> Brain Res. Dev. Brain Res. 130, 41-51 (2001).</li><li>Mao, L., Wang, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upregulation+of+preprodynorphin+and+preproenkephalin+mRNA+expression+by+selective+activation+of+group+I+metabotropic+glutamate+receptors+in+characterized+primary+cultures+of+rat+striatal+neurons.">Upregulation of preprodynorphin and preproenkephalin mRNA expression by selective activation of group I metabotropic glutamate receptors in characterized primary cultures of rat striatal neurons.</a> Brain Res. Mol. Brain Res. 86, 125-137 (2001).</li><li>Oorschot, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+fluorodeoxyuridine+on+neurons+and+non-neuronal+cells+in+cerebral+explants.">Effect of fluorodeoxyuridine on neurons and non-neuronal cells in cerebral explants.</a> Exp. Brain Res. 78, 132-138 (1989).</li><li>Price, P. J., Brewer, G. J., Federoff, S., Richardson, A. <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+media+for+neural+cell+cultures.">Serum -free media for neural cell cultures.</a> Protocols for Neural Cell Culture. , (2001).</li><li>Shen, J., Watanabe, S., Kaneko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+dissociation+with+papain+reduces+the+density+of+cGMP-activated+channels+of+the+retinal+rod.">Cell dissociation with papain reduces the density of cGMP-activated channels of the retinal rod.</a> Jpn. J. Physiol. 45, 151-164 (1995).</li><li>Stratmann, G., Sall, J. W., May, L. D., Loepke, A. W., Lee, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+anesthetic+properties:+The+effects+of+isoflurane+on+brain+cell+death,+neurogenesis+and+long-term+neurocognitive+function.">Beyond anesthetic properties: The effects of isoflurane on brain cell death, neurogenesis and long-term neurocognitive function.</a> Anesthesia and Analgesia. 110, 431-437 (2009).</li><li>Wallace, T. L., Johnson, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytosine+arabinoside+kills+postmitotic+neurons:+evidence+that+deoxycytidine+may+have+a+role+in+neuronal+survival+that+is+independent+of+DNA+synthesis.">Cytosine arabinoside kills postmitotic neurons: evidence that deoxycytidine may have a role in neuronal survival that is independent of DNA synthesis.</a> J. Neurosci. 9, 115-124 (1989).</li></ol>

Hippocampale kulturer er blevet anvendt i molekylærbiologi i mere end 20 år. Mens der i princippet kan neuronale kulturer fremstilles af en del af hjernen, har hippocampale kulturer vist sig at være den mest populære på grund af den relativt enkle opbygning af nerve-cellepopulation i hippocampus 7. Hippocampus kulturer er typisk fremstillet af senfase fostervæv. Dette væv er lettere at dissociere og indeholder færre gliaceller, end modent hjernevæv 1. Isolering af hippocampale neuroner fra...

<table border="1"><tbody><tr><td> Navn reagens </td><td> Vendor </td><td> Katalognummer </td></tr><tr><td> Rotte Hale Collagen 1 </td><td> BD Biosciences </td><td> 354236 </td></tr><tr><td> Poly-D-lysinopløsningen </td><td> Chemicon </td><td> A-003-E </td></tr><tr><td> Hanks balancerede saltopløsning </td><td> Invitrogen </td><td> 14175-095 </td></tr><tr><td> Trypsin-opløsning (1x) 0,25%, flydende </td><td> Invitrogen </td><td> 15050-065 </td></tr><tr><td> Neurobasal medium (1X) væske </td><td> Invitrogen </td><td> 21103-049 </td></tr><tr><td> B27 Supplement (50X) væske </td><td> Invitrogen </td><td> 17504-044 </td></tr><tr><td> L-Glutamin 200 mM (100X) væske </td><td> Invitrogen </td><td> 25030-149 </td></tr><tr><td> Penicillin (10.000 enheder / ml) / streptomycin (10.000 ug / ml) </td><td> Invitrogen </td><td> 15140-148 </td></tr><tr><td> HI-Donor Horse Serum </td><td> Atlanta Biologicals </td><td> S12150H </td></tr></tbody></table>

isolation and culture of hippocampal neurons from prenatal mice

Primære kulturer af rotte-og murin hippocampale neuroner er almindeligt anvendt til at afsløre cellulære mekanismer neurobiologi. Ved at isolere og voksende individuelle neuroner, er forskere i stand til at analysere egenskaber i forbindelse med cellulære handel cellestruktur og individuelle protein lokalisering ved hjælp af en række af biokemiske teknikker. Resultater fra sådanne forsøg er afgørende for at teste teorier der vedrører den neurale basis for hukommelse og indlæring. Imidlertid er utvetydige resultater fra disse former af eksperimenter baseret på evnen til at vokse neuronale kulturer med minimal kontaminering fra andre hjerneregioner celletyper. I denne protokol, bruger vi bestemte medier, designet til neuron vækst og omhyggelig dissektion af embryonale hippocampalt væv til at optimere væksten af ​​sunde neuroner og samtidig minimere forurenende celletyper (dvs. astrocytter). Embryonisk mus hippocampalt væv kan være vanskeligere at isolere end tilsvarende gnaver væv på grund af størrelsen af ​​prøven for dissection. Vi viser detaljerede dissektion teknikker hippocampus fra embryonale dag 19 (E19) museunger. Når hippocampalt væv isoleres, er skånsom dissociation af neuronale celler opnået med en fortyndet koncentration af trypsin og mekanisk sprængning konstrueret til at adskille celler fra bindevævet samtidig med minimal beskadigelse af de enkelte celler. En detaljeret beskrivelse af, hvordan man forbereder pipetter, der anvendes i forstyrrelsen er inkluderet. Optimale plettering tætheder er fastsat for immuno-fluorescens-protokoller til at maksimere succes cellekultur. Protokollen tilvejebringer en hurtig (ca. 2 timer) og effektiv teknik til dyrkning af neuronale celler fra mus hippocampalt væv.

Watch this Scientific Journal Video about Isolation and Culture of Hippocampal Neurons from Prenatal Mice at JoVE.com

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Vi en protokol til dyrkning af højrenset hippocampale neuroner fra prænatal musehjerner uden anvendelse af en føder gliacelle lag.

Isolering og dyrkning af hippocampus neuroner fra Prænatal Mus

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

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