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 hjelp i utarbeidelsen av manuskriptet. Dette arbeidet ble støttet av NIH 2RO1NS033661 (MWW).

1. Sett opp Før Harvest <ol><li> For å generere prenatal valper for nevron høst, planlegge avl mellom voksne mus 19 dager før den dagen neuron isolasjon. (C57BL / 6 mus aldre 2-8 måneder ble brukt i parringer med henblikk på å utvikle denne protokollen). Vellykket parring kan bekreftes ved påvisning av en vaginal plugg i den kvinnelige, hjertebank eller visuell bekreftelse av svangerskapet. </li><li> Dagen før neuron isolasjon: <ol class="ualpha"><li> For immunfluorescens applikasjoner, jakke glass D...

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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>

Hippocampus kulturer har blitt brukt i molekylær biologi i mer enn 20 år. Mens i prinsippet kan nevrale kulturer gjøres fra hvilken som helst del av hjernen, har hippocampus kulturer vist seg å være den mest populære på grunn av den relativt enkle arkitekturen av nerve cellen befolkningen i hippocampus 7. Hippocampus kulturer er vanligvis laget av sent stadium embryonale vev. Dette vevet er lettere å distansere og inneholder færre gliacellene enn gjør moden hjernevev en. Isolering av hipp...

<table border="1"><tbody><tr><td> Navn på Reagens </td><td> Selger </td><td> Catalog Number </td></tr><tr><td> Rat Tail Kollagen 1 </td><td> BD Biosciences </td><td> 354236 </td></tr><tr><td> Poly-D-lysin Solution </td><td> Chemicon </td><td> A-003-E </td></tr><tr><td> Hanks Balansert Salt Solution </td><td> Invitrogen </td><td> 14175-095 </td></tr><tr><td> Trypsin Solution (1X) 0,25%, flytende </td><td> Invitrogen </td><td> 15050-065 </td></tr><tr><td> NeuroBasal Medium (1X) flytende </td><td> Invitrogen </td><td> 21103-049 </td></tr><tr><td> B27 Supplement (50X) flytende </td><td> Invitrogen </td><td> 17504-044 </td></tr><tr><td> L-Glutamin 200 mm (100X) flytende </td><td> Invitrogen </td><td> 25030-149 </td></tr><tr><td> Penicillin (10.000 enheter / ml) / Streptomycin (10.000 mikrogram / 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 av rotter og murine hippocampus nevroner er mye brukt til å avsløre cellulære mekanismene i nevrobiologi. Ved å isolere og voksende enkelte nevroner, forskerne er i stand til å analysere egenskaper knyttet til mobilnettet menneskehandel, cellestruktur og individuell protein lokalisering ved hjelp av en rekke biokjemiske teknikker. Resultater fra slike eksperimenter er kritiske for å teste teorier adressering det nevrale grunnlaget for hukommelse og læring. Imidlertid er entydige resultater fra disse formene for eksperimentene betinget av evnen til å vokse nevrale kulturer med minimum forurensning av andre hjernen celletyper. I denne protokollen, bruker vi spesifikke mediet er laget for neuron vekst og forsiktig disseksjon av embryonale hippocampus vev for å optimalisere veksten av sunne nevroner samtidig som forurensende celletyper (dvs. astrocytes). Embryonale mus hippocampus vev kan være vanskeligere å isolere enn tilsvarende gnager vev på grunn av størrelsen av utvalget for dissection. Vi viser detaljerte disseksjon teknikker av hippocampus fra embryonale dag 19 (E19) mus valper. Når hippocampus vev er isolert, er skånsom dissosiasjon av nevrale celler oppnådd med en fortynnet konsentrasjon av trypsin og mekanisk avbrudd for å separere celler fra bindevev samtidig som det gir minst skade på individuelle celler. En detaljert beskrivelse av hvordan å forberede pipetter skal brukes ved forstyrrelser er inkludert. Optimale plating tettheter er gitt for immuno-fluorescens protokoller for å maksimere vellykket cellekultur. Protokollen gir en rask (ca. 2 timer) og effektiv teknikk for kulturen i nevrale celler fra mus hippocampus vev.

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 tilbyr en protokoll for kultur høyt renset hippocampus nevroner fra prenatal mus hjerner uten bruk av en mater glial celle laget.

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