Name: bilaminar co-culture of primary rat cortical neurons and glia
Uploaded: 2011-11-12T14:00:00
Description: Watch this Scientific Journal Video about Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia at JoVE.com

Forfatterne takker forrige laboratorium medlemmer som har bidratt til forbedringer av denne protokollen og NIH for støtte gjennom årene (DA19808 og DA15014 til OM). Anna Abt 1 er en kar av &quot;Tverrfaglig og translasjonsforskning Opplæring i neuroAIDS&quot; (T32-MH078795), derfor ble dette arbeidet støttet delvis av National Institutes of Health i henhold Ruth L. Kirschstein National Research Service Award 5T32MH079785.

1. Gliaceller Dissection (~ 2 uker før plating nevroner) <ol><li> Å forberede seg til disseksjon sted sterile verktøy i 70% etanol, tilsett 4 mL kaldt disseksjon medium til 60 mm retter (ett fat per hjernen), og plassere 2,5% trypsin og DNase på is for å tine (se detaljer om kirurgisk verktøy i tabell VI ). </li><li> Bruk stor saks til å halshogge 2-4 dager gamle valper og sted hoder i en 100 mm steril parabolen. </li><li> Bruk medium saks å lage en midtlinje skjære gjennom huden og trekke tilbake huden å av...

<ol>
	<li>Cook, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+chemokines:+regulation+of+fractalkine/CX3CL1+homeostasis+by+SDF/CXCL12+in+cortical+neurons.">Interactions between chemokines: regulation of fractalkine/CX3CL1 homeostasis by SDF/CXCL12 in cortical neurons.</a> J. Biol. Chem. 285, 10563-10563 (2010).</li><li>Nicolai, J., Burbassi, S., Rubin, J., Meucci, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCL12+inhibits+expression+of+the+NMDA+receptor's+NR2B+subunit+through+a+histone+deacetylase-dependent+pathway+contributing+to+neuronal+survival.">CXCL12 inhibits expression of the NMDA receptor's NR2B subunit through a histone deacetylase-dependent pathway contributing to neuronal survival.</a> Cell. Death. Dis. 1, e33-e33 (2010).</li><li>Sengupta, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphine+increases+brain+levels+of+ferritin+heavy+chain+leading+to+inhibition+of+CXCR4-mediated+survival+signaling+in+neurons.">Morphine increases brain levels of ferritin heavy chain leading to inhibition of CXCR4-mediated survival signaling in neurons.</a> J. Neurosci. 29, 2534-2544 (2009).</li><li>Meucci, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+regulate+hippocampal+neuronal+signaling+and+gp120+neurotoxicity.">Chemokines regulate hippocampal neuronal signaling and gp120 neurotoxicity.</a> Proc. Natl. Acad. Sci. U. S. A. 95, 14500-14500 (1998).</li><li>Khan, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+neuronal+P53+activity+by+CXCR+4.">Regulation of neuronal P53 activity by CXCR 4.</a> Mol. Cell. Neurosci. 30, 58-66 (2005).</li><li>Patel, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+neuronal+CXCR4+by+the+micro-opioid+agonist+DAMGO.">Modulation of neuronal CXCR4 by the micro-opioid agonist DAMGO.</a> J. Neurovirol. 12, 492-500 (2006).</li><li>Shimizu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+transcription+factor+E2F1+in+CXCR4-mediated+neurotoxicity+and+HIV+neuropathology.">Role of the transcription factor E2F1 in CXCR4-mediated neurotoxicity and HIV neuropathology.</a> Neurobiol. Dis. 25, 17-26 (2007).</li><li>Khan, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemokine+receptor+CXCR4+regulates+cell-cycle+proteins+in+neurons.">The chemokine receptor CXCR4 regulates cell-cycle proteins in neurons.</a> J. Neurovirol. 9, 300-314 (2003).</li><li>Khan, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemokine+CXCL12+promotes+survival+of+postmitotic+neurons+by+regulating+Rb+protein.">The chemokine CXCL12 promotes survival of postmitotic neurons by regulating Rb protein.</a> Cell. Death. Differ. 15, 1663-1672 (2008).</li><li>Khan, M. Z., Vaidya, A., Meucci, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCL12-mediated+regulation+of+ANP32A/Lanp,+a+component+of+the+inhibitor+of+histone+acetyl+transferase+(INHAT)+complex,+in+cortical+neurons.">CXCL12-mediated regulation of ANP32A/Lanp, a component of the inhibitor of histone acetyl transferase (INHAT) complex, in cortical neurons.</a> J. Neuroimmune. Pharmacol. 6, 163-170 (2011).</li><li>Dotti, C. G., Sullivan, C. A., Banker, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+establishment+of+polarity+by+hippocampal+neurons+in+culture.">The establishment of polarity by hippocampal neurons in culture.</a> J. Neurosci. 8, 1454-1468 (1988).</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>Goslin, K., Banker, G., Goslin, K., 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=.">.</a> Culturing Nerve Cells. , 339-370 (1998).</li><li>D'Ambrosio, J., Fatatis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoblasts+modulate+Ca2++signaling+in+bone-metastatic+prostate+and+breast+cancer+cells.">Osteoblasts modulate Ca2+ signaling in bone-metastatic prostate and breast cancer cells.</a> Clin. Exp. Metastasis. 26, 955-964 (2009).</li></ol>

Denne protokollen gir en metode for dyrking rotte primære kortikale nevroner i nærvær av gliaceller celler, samtidig som nervecellene skal være lett isolert for eksperimentell analyse. Den gliaceller støtte utviklingen av en sunn neuronal fenotype, mens også modulerende nevrale responser til eksperimentelle behandlinger i en fysiologisk relevant måte. I tillegg, ved passaging den primære gliaceller før dyrking med nevroner denne cellen befolkning blir selektivt anriket i astrocytes, og dermed hindre inflammator...

<table border="1">
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Concentration</th>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>16 mM</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>22 mM</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>0.22 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>pH</td>
			<td>7.4</td>
		</tr>
		<tr>
			<td>Osmolarity</td>
			<td>310&plusmn;10 mOsm</td>
		</tr>
	</tbody>
</table>
Table 1. Dissection Medium.
<table border="1">
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Concentration</th>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>90%</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>50 &mu;g/mL</td>
		</tr>
	</tbody>
</table>
Tabe 2. Glia Plating Medium.
<table border="1">
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Concentration</th>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>90%</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>10%</td>
		</tr>
	</tbody>
</table>
Table 3. Neuron Plating Medium.
<table>
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Concentration</th>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>98%</td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>1M HEPES</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>Ovalbumin</td>
			<td>50 mg/100mL</td>
		</tr>
	</tbody>
</table>
Table 4. N2 Medium.
<table border="1">
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Concentration</th>
		</tr>
		<tr>
			<td>Boric Acid</td>
			<td>50 mM</td>
		</tr>
		<tr>
			<td>Sodium Borate</td>
			<td>12.5 mM</td>
		</tr>
	</tbody>
</table>
Table 5. Borate Buffer (for poly-lysine).
<table border="1">
	<tbody>
		<tr>
			<th>
				Reagent</th>
			<th>
				Company</th>
			<th>
				Catalogue number</th>
		</tr>
		<tr>
			<td>High glucose Dulbecco's Modified Eagle 
			Medium (DMEM)</td>
			<td>Invitrogen</td>
			<td>11995-073</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS), Heat-inactivated</td>
			<td>Hyclone</td>
			<td>26400-044</td>
		</tr>
		<tr>
			<td>Horse Serum, Heat-inactivated</td>
			<td>Hyclone</td>
			<td>H1138</td>
		</tr>
		<tr>
			<td>Gentamicin (50mg/mL)</td>
			<td>Invitrogen</td>
			<td>15750-060</td>
		</tr>
		<tr>
			<td>N2 Supplement (100x)</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
		</tr>
		<tr>
			<td>HEPES buffer solution</td>
			<td>Invitrogen</td>
			<td>15630-080</td>
		</tr>
		<tr>
			<td>Albumin from chicken egg white, Grade VI 
			(Ovalbumin)</td>
			<td>Sigma-Aldrich</td>
			<td>A2512</td>
		</tr>
		<tr>
			<td>2.5% Trypsin</td>
			<td>Invitrogen</td>
			<td>15090-046</td>
		</tr>
		<tr>
			<td>0.5% Trypsin-EDTA (10X)</td>
			<td>Invitrogen</td>
			<td>15400-054</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas 
			(DNase)</td>
			<td>Sigma-Aldrich</td>
			<td>D-5025</td>
		</tr>
		<tr>
			<td>Paraplast</td>
			<td>Fisher</td>
			<td>12-646-106</td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P1274</td>
		</tr>
		<tr>
			<td>Cytosine-&beta;-D-arabinofuranoside hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>C6645</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>Leica ZOOM 2000</td>
		</tr>
		<tr>
			<td>Cover glasses, Circles, 15 mm, Thickness 
			0.13-0.17 mm</td>
			<td>Carolina</td>
			<td>633031</td>
		</tr>
		<tr>
			<td>Thermanox sheets</td>
			<td>Grace BioLabs</td>
			<td>HS4550</td>
		</tr>
		<tr>
			<td>Large forceps</td>
			<td>Biomedical 
			Research 
			Instruments</td>
			<td>70-4000</td>
		</tr>
		<tr>
			<td>Fine-tipped No.5 forceps</td>
			<td>Fine Science 
			Tools</td>
			<td>91150-20</td>
		</tr>
		<tr>
			<td>Pattern No.1 forceps</td>
			<td>Biomedical 
			Research</td>
			<td>10-1400</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Instruments</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scissors, straight, sharp-blunt</td>
			<td>Biomedical 
			Research 
			Instruments</td>
			<td>28-1435</td>
		</tr>
		<tr>
			<td>Micro Dissecting scissors</td>
			<td>Biomedical 
			Research 
			Instruments</td>
			<td>11-2070</td>
		</tr>
		<tr>
			<td>Micro Dissecting Curved scissors</td>
			<td>Biomedical 
			Research 
			Instruments</td>
			<td>11-1395</td>
		</tr>
	</tbody>
</table>

bilaminar co-culture of primary rat cortical neurons and glia

Denne videoen vil guide deg gjennom prosessen med dyrking rotte kortikale nevroner i nærvær av en glial mater lag, et system kjent som en bilaminar eller co-kulturen modell. Dette systemet er egnet for en rekke eksperimentelle behov som krever enten et glass eller plast vekst substrat, og kan også brukes til kultur for andre typer nerveceller. Rat kortikale nevroner hentet fra slutten av fosterstadiet (E17) er belagt på glass Dekkglass eller vev kultur retter overfor en mater lag av gliaceller vokst på oppvasken eller plast Dekkglass (kjent som Thermanox), henholdsvis. Valget mellom de to konfigurasjoner avhenger av den aktuelle eksperimentelle teknikken som brukes, noe som kan kreve, eller ikke, er at nervecellene vokst på glass (f.eks kalsium bildebehandling versus Western blot). Den glial mater lag, en astroglia-beriket sekundære kultur blandet gliaceller, er separat forberedt fra cortices av nyfødte rotteunger (P2-4) før neuronaldisseksjon. En stor fordel med denne kulturen system i forhold til en kultur av nerveceller er bare støtte av neuronal vekst, overlevelse, og differensiering leveres av trofiske faktorer skilles fra glial mater laget, som mer nøyaktig ligner hjernen miljøet in vivo. Videre kan co-kulturen brukes til å studere neuronal-glial interaksjoner 1. Samtidig er gliaceller forurensning i nevronale lag hindres av ulike virkemidler (low density kultur, tillegg av mitotisk hemmere, mangel på serum og bruk av optimalisert kultur medium) fører til en nesten ren nevrale lag, kan sammenlignes med andre etablerte metoder 1 -3. Nevroner kan lett skilles fra glial lag når som helst under kultur og brukes til ulike eksperimentelle applikasjoner som spenner fra 4 elektrofysiologi, cellulær og molekylær biologi 5-8, biokjemi 5, bildebehandling og microscopy 4,6,7,9,10. Den primære nevroner utvide axoner og dendritter å danne funksjonelle synapser 11, en ​​prosess som ikke er observert i neuronal cellelinjer, selv om noen cellelinjer gjør utvide prosesser. En detaljert protokoll av dyrkning rotte hippocampus nevroner bruker denne co-kulturen system har blitt beskrevet tidligere 4,12,13. Her har vi detalj en modifisert protokoll egnet for kortikale nevroner. Som omtrent 20x10 6 celler er gjenvunnet fra hver rotte embryo, er denne metoden spesielt nyttig for eksperimenter som krever et stort antall nevroner (men ikke bekymret for en svært homogen neuronal befolkningen). Utarbeidelse av nevroner og gliaceller må planlegges i en tid-spesifikk måte. Vi vil gi trinn-for-steg protokoll for dyrking rotte kortikale nevroner samt dyrking gliaceller å støtte nervecellene.

Watch this Scientific Journal Video about Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia at JoVE.com

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

Her tilbyr vi en protokoll for dyrking rotte kortikale nevroner i nærvær av en glial mater lag. Den dyrkede nerveceller etablere polaritet og skape synapser, og kan skilles fra gliaceller for bruk i ulike programmer, som elektrofysiologi, kalsium bildebehandling, celle overlevelse analyser, immunocytochemistry, og RNA / DNA / protein isolasjon.

Bilaminar Co-kultur Primary Rat kortikale nevroner og gliaceller

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

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