Name: spinal cord neurons isolation and culture from neonatal mice
Uploaded: 2017-07-11T15:00:00
Description: Watch this Scientific Journal Video about Spinal Cord Neurons Isolation and Culture from Neonatal Mice at JoVE.com

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Pleje og behandling af dyr i denne procedure blev udført i overensstemmelse med retningslinjerne i Instituttet for Dyrpleje og Brug ved University of Colorado. 1. Forberedelse af løsninger <ol><li> Forbered og opbevar alle opløsninger ved passende temperaturer, som vist i tabel 1 . </li></ol> 2. Coating Wells og dias BEMÆRK: Neuroner holder sig ikke godt til plast- eller glasflader. <ol><li> En dag før isolering a...

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J., et al. <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 (5), 567-576 (1993).</li><li>Fang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+preconditioning+protects+against+spinal+cord+ischemia-reperfusion+injury+in+rabbits+by+attenuating+blood+spinal+cord+barrier+disruption.">Ischemic preconditioning protects against spinal cord ischemia-reperfusion injury in rabbits by attenuating blood spinal cord barrier disruption.</a> Int J Mol Sci. 14 (5), 10343-10354 (2013).</li><li>Su, M., Zhong, W., Ren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose-dependent+protection+of+reseveratrol+against+spinal+cord+ischemic-reperfusion+injury+in+rats.">Dose-dependent protection of reseveratrol against spinal cord ischemic-reperfusion injury in rats.</a> Trop J Pharm Res. 15 (6), 1225-1233 (2016).</li><li>Haapanen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+ischemic+preconditioning+protects+the+spinal+cord+against+ischemic+insult:+An+experimental+study+in+a+porcine+model.">Remote ischemic preconditioning protects the spinal cord against ischemic insult: An experimental study in a porcine model.</a> J Thorac Cardiovasc Surg. 151 (3), 777-785 (2016).</li><li>Conrad, M. 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Denne teknik muliggør den pålidelige kultur af rygmarvsneuroner. Når færdigheder i teknikken er nået, tager det cirka 3,5 timer at afslutte. Vi har været i stand til at udføre isolationen af ​​neuroner fra 2 separate kuldarter (16 mus totalt) om cirka 4 timer. Nøglefasen i gennemførlighed er i stand til at uddrage spinalkablerne effektivt fra musene. Udbyttet tillader plating af flere brønde og for evnen til at teste neuronerne under forskellige betingelser. Vi har været i stand til at behandle neuronerne...

<html> Forståelse af rygmarvspatologi kræver brug af forskellige modeller, både på makroskopiske og mikroskopiske niveauer. Store og små dyremodeller 1 , 2 , 3 anvendes til in vivo undersøgelser af rygmarvs sygdom og skade. Mens undersøgelsen af ​​disse problemer in vivo har sine fordele, er rygmarvanalysen begrænset til hele rygmarvshomogenat eller til vævssektioner 4 . Dette skaber en vis tvetydighed, når man forsøger at isolere specifikke svar og mål i rygmarven blandt sine residente neuroner og omgivende glia. Den stigende tilgængelighed af genetisk manipulerede mus giver mulighed for mere detaljerede undersøgelser af biologien på cellulære og molekylære niveauer. Således anvendes en neonatal musemodel her, hvilket muliggør undersøgelse af de unikke egenskaber og biologi af rygmarvsneuroner in vitro . Ent &quot;&gt; Isolering og vedligeholdelse af neuroner in vitro er ikke særlig ligetil. Der er en relativ overflod af teknikker til neuronisolering fra det kortikale væv hos voksne gnavere, der synes at resultere i et betydeligt antal isolerede neuroner ( dvs. millioner) 5 , 6 , 7. I modsætning hertil er udbyttet af neuroner fra rygmarvsvæv lavere 8 , 9 , 10 , dels på grund af den mindre vævemasse. Desuden er der hos mus en relativ sparsomhed af teknikker til isolering af neonatal Rygmarvsneuroner, og eksisterende metoder er begrænset af lavere neuronudbytter ( dvs. hundredvis) 9 eller krævende og ressourcetunge teknikker, der kræver isolering af embryonale mus 10 . I denne protokol, viBrug en teknik, der muliggør den omkostningseffektive isolering af et betydeligt antal neuroner fra neonatale muses rygmarv. Som det er almindeligt i tidligere offentliggjorte teknikker bruger vi papain som en enzymatisk protease, der tillader frigivelse af neuroner fra rygmarvvæv 5 , 6 . Derudover bruger vi en densitetsgradient til raffineret celleseparation, som tidligere har vist sig at være effektiv 6 , 10 . Medens mediet, hvori cellerne inkuberes, kan variere i vores erfaring og som tidligere offentliggjort 11 , har tillæg med frisk B27-kulturmediumtilskud vist sig at være kritisk for neuronlevetiden. Neuronerne er typisk levedygtige i op til 10 dage, hvilket gør det muligt at udføre behandlingen.

<table border="0" cellpadding="0" cellspacing="0" width="1357">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Hibernate A Medium - 500 mL</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td style="width:107px;">A1247501</td>
			<td style="width:805px;">https://www.thermofisher.com/order/catalog/product/A1247501</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hibernate A Minus Calcium - 500 mL</td>
			<td style="width:116px;">Brainbits</td>
			<td>HA-Ca</td>
			<td>http://www.brainbitsllc.com/hibernate-a-minus-calcium/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutamax 100X - 100 mL</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td>35050061</td>
			<td>https://www.thermofisher.com/order/catalog/product/35050079</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B27 Supplement 50X - 10 mL</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td>17504044</td>
			<td>https://www.thermofisher.com/order/catalog/product/17504044</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Papain, Lyophilized - 100 mg</td>
			<td style="width:116px;">Worthington</td>
			<td>LS003119</td>
			<td>http://www.worthington-biochem.com/pap/cat.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal A Medium - 500 mL</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td>10888022</td>
			<td>https://www.thermofisher.com/order/catalog/product/10888022</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td>15140122</td>
			<td>https://www.thermofisher.com/order/catalog/product/15140122</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-D-lysine hydrobromide - 5 mg</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>P6407-5MG</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/p6407?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Laminin - 1 mg</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td>23017015</td>
			<td>https://www.thermofisher.com/order/catalog/product/23017015</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue - 20 mL</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>T8154-20ML</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/t8154?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OptiPrep Density Gradient Medium - 250 mL</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>D1556-250ML</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/d1556?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dichlorodimethylsilane (DMDCS, Sigma Silicoat)</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>440272-100ML</td>
			<td>http://www.sigmaaldrich.com/catalog/product/aldrich/440272?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>288306-1L</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sial/288306?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Glass Pippette - 9&#34;</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td style="width:107px;">13-678-20C</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/cls7095d9?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipette bulb - 5 mL</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>Z186678-3EA</td>
			<td>http://www.sigmaaldrich.com/catalog/product/aldrich/z186678?lang=en&#38;region=US&#38;cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold10-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BRAND&#174;&#160;Petri dish, glass - 60x15 mm</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>BR455717-10EA</td>
			<td>http://www.sigmaaldrich.com/catalog/product/aldrich/br455717?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile 24 Well Cell Culture Plate</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>M8812-100EA</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/m8812?lang=en&#38;region=US</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Hausser Hemacytometer (glass counting chamber)</td>
			<td style="width:116px;">Fischer Scientific</td>
			<td>02-671-6</td>
			<td>https://www.fishersci.com/shop/products/hausser-bright-line-phase-hemacytometer-hemacytometer/026716</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Slides - 12 mm sterile cover glass - uncoated</td>
			<td>Neuvitro</td>
			<td>GG-12-1.5-Pre</td>
			<td>http://www.neuvitro.com/german-coverslip-12mm-diameter.htm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NeuN Rabbit Monoclonal Antibody - 100 &#181;L</td>
			<td style="width:116px;">Abcam</td>
			<td>ab177487</td>
			<td>After fixing in paraformaldehyde and blocking with 5% BSA, cells on a 12 mm coverslip were incubated in the antibody diluded to 1:200 for 18 hours in 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MAP-2 Mouse Monoclonal Antibody - 50 &#181;L</td>
			<td style="width:116px;">Abcam</td>
			<td>ab11267</td>
			<td>After fixing in paraformaldehyde and blocking with 5% BSA, cells on a 12 mm coverslip were incubated in the antibody diluded to 1:500 for 18 hours in 4 &#176;C</td>
		</tr>
	</tbody>
</table>

spinal cord neurons isolation and culture from neonatal mice

Vi præsenterer en protokol for isolering og kultur af rygmarvsneuroner. Neuronerne opnås fra neonatal C57BL / 6 mus og isoleres på postnatal dag 1-3. Et musestøv, der normalt er 4-10 pupper født fra et avlspar, samles til et eksperiment, og spinalkabler opsamles individuelt fra hver mus efter eutanasi med isofluran. Ryggsøjlen er dissekeret og derefter frigives rygmarven fra søjlen. Spinalkablerne bliver derefter hakket for at forøge overføringsareal for en enzymatisk protease, der tillader, at neuronerne og andre celler frigives fra vævet. Trituration bruges derefter til at frigive cellerne i opløsning. Denne opløsning fraktioneres efterfølgende i en densitetsgradient for at adskille de forskellige celler i opløsning, hvilket tillader at neuroner isoleres. Ca. 1-2,5 x 106 neuroner kan isoleres fra en kuldgruppe. Neuronerne er så podet på brønde overtrukket med klæbende factoRs, der giver mulighed for ordentlig vækst og modning. Neuronerne tager ca. 7 dage at nå modenhed i vækst- og kulturmediet og kan derefter anvendes til behandling og analyse.

Ved hjælp af denne teknik muliggør et enkelt kuld (4-10 pupper) isolering af 1-2,5 10 6 neuroner, der er egnede til såning på kulturplader. Typisk frøes 4-8 brønde ved den ovenfor nævnte koncentration ( dvs. 300.000 celler / ml). Figur 3 demonstrerer udseendet af neuroner ved denne koncentration efter en uge i kultur ved lav- ( a ) og høj- ( b ) forstørrelseslysmikroskopi. Vi har dog også været...

Watch this Scientific Journal Video about Spinal Cord Neurons Isolation and Culture from Neonatal Mice at JoVE.com

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

Denne undersøgelse præsenterer en teknik til isolering af neuroner fra WT neonatale mus. Det kræver en forsigtig dissektion af rygmarven fra den neonatale mus, efterfulgt af adskillelsen af ​​neuroner fra rygmarvvævet gennem mekanisk og enzymatisk spaltning.

Spinal Cord Neurons Isolation og Culture fra Neonatal Mus

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

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