Name: isolation and culture of dissociated sensory neurons from chick embryos
Uploaded: 2014-09-24T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos at JoVE.com

We willen graag Alison Philbrook, Belinda Barbagallo en Michael Francis bedanken voor inzichtelijke reacties op dit papier. Onderzoek gemeld in deze publicatie werd ondersteund door een R15 AREA award 1R15NS070172-01A1 toegekend aan MLL.

1 Coverslip Voorbereiding: Acid Wash en Bake <ol><li> Minstens 2 dagen voor dissectie (stap 2), begint de volgende stappen uit om dekglaasjes bereiden. Voer de zure wasstap om oliën en grondig schoon dekglaasjes verwijderen. </li><li> Load dekglaasjes in porseleinen houder die verticaal positioneert dekglaasjes en voorkomt dat ze elkaar raken. Dompel houder en dekglaasjes in glas histologie container gevuld met 2 M zoutzuur (HCl). Als alternatief, als porselein houder niet beschikbaar, verspreid ~ 20 dekglaasjes hori...

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Hier presenteren wij gedetailleerde protocollen voor het isoleren en kweken los sensorische neuronen van een kippenembryo. Deze procedure genereert een verrijkte populatie van stevig groeiende neuronen in vitro 7,8,10,16. Vele cellulaire en moleculaire technieken kunnen worden toegepast op deze gekweekte neuronen, waaronder immunocytochemie, die hier wordt beschreven. Dit protocol werd onlangs gebruikt om de intensiteit van immunologisch actieve integrinen sensorische groei kegels 8,16 kwa...

Neuronen zijn complexe cellen die informatie essentieel is voor een verscheidenheid aan functies, waaronder sensatie, visie, motoriek, leren en geheugen uit te voeren. Unieke van andere celtypen, neuronen verlengen arm-achtige processen axonen, essentiële neurale snelwegen communicatie vormen. Tijdens de ontwikkeling van gespecialiseerde compartimenten zich aan de uiteinden van de groeiende axonen, de zogenaamde groei kegels, navigeren door een concert van extracellulaire signalen naar de axon naar de juiste bestemming te leiden. De ingewikkelde moleculaire mechanismen die groeikegel navigatie grondslag liggen zijn niet volledig begrepen. Om beter te begrijpen van deze mechanismen, hebben de onderzoekers gebruikt celcultuur modellen voor neuronen in een gedefinieerde en vereenvoudigde in vitro-omgeving te bestuderen. Studeren neuronen in cultuur 1 heeft geleid tot aanzienlijke vooruitgang in ons begrip van de neuronale celbiologie waaronder: neuronale differentiatie 2, cytoskelet dynamiek, endocytose en mensenhandel, dendrietregulatie 3,4, axonale regeneratie 5, en klinische aandoeningen zoals neuropathieën 6. Bovendien gekweekte neuronen zeer vatbaar voor diverse onderzoekstechnieken zoals immunocytochemie, celoppervlak co-immunoprecipitatie, Western blot, transfectie, RNAi en levende imaging zoals timelapse analyse van groeikegel motiliteit. Aldus kweken van primaire neuronen is een krachtige aanpak talrijke aspecten van de celbiologie van neuronen helderen. De celcultuur model biedt onderzoekers met gedetailleerde controle over de milieu-omstandigheden en variabelen. Bijvoorbeeld kan het substraat waarop neuronen uitgeplaat (en groeien op) gemakkelijk worden gemanipuleerd. Hier geven we gedetailleerde instructies voor het genereren van twee verschillende substraten, een met een lage concentratie laminine-1 en de andere met verzadigende concentraties van laminine-1. Verrassenderwijs kunnen verschillende concentraties van hetzelfde molecuul dramatische effectende interne toestand van neuronen en hun celoppervlak samenstelling. Bijvoorbeeld, intracellulaire niveaus van cAMP en maaivelden integrines significant verschillend in neuronen uitgeplaat op beide substraten 7,8. Aanvullende studies hebben aangetoond dat andere moleculen, waaronder fibronectine en chondroïtinesulfaat proteoglycanen, effect de expressie van celoppervlak moleculen en neuronale motiliteit 7-11. Bovendien oplosbare moleculen zoals neurotrofinen en neurotropins ook gevolgen celmembraan preparaat en neuronale beweeglijkheid 12-16 en kan gemakkelijk en nauwkeurig gemanipuleerd in een cel cultuurmodel. Hier beschrijven we methoden om te isoleren en cultuur los sensorische neuronen van kippenembryo&#39;s. Deze procedure is gebruikt om belangrijke doorbraken in neurobiologie, inclusief axon uitgroei 5,7,8,10,11,16-21 maken en is een modificatie van een procedure die tot ganglioncellen 22 isoleren. Er zijnverscheidene voordelen aan deze aanpak. Ten eerste zijn veel kenmerken van chick dorsale wortel ganglion (DRG) ontwikkeling goed gekarakteriseerd, waaronder de termijn voor de geboorte, axon extensie, en eiwit expressie profielen 2,23-28, waardoor het een leerzame basis waarop te bouwen informatieve in vitro experimenten. Ten tweede, gedissocieerd neuronale kweken kan de onderzoeker directer bestuderen neuronen vergelijking met alternatieve benaderingen waarbij intacte DRG-explantaten (welke neuronen en niet-neuronale cellen bevatten) en / of gemengde kweken die zowel gedissocieerde neuronen en niet-neuronale cellen. Ten derde, de hier beschreven procedure is eenvoudig, goedkoop en vatbaar voor studenten. Daarom kan deze techniek worden gebruikt voor onderzoek en voor onderwijsdoeleinden. Bovendien moet kleine wijzigingen van dit protocol snelle, hoge opbrengst zuivering van neuronen uit andere dan DRGs bronnen toestaan. Bijvoorbeeld kan deze procedure worden aangepast om neuronaal verschaffen Enriched kweken van andere weefsels zoals embryonale voorhersenen of ruggenmerg. Immunocytochemie protocollen zijn geoptimaliseerd voor deze gedissocieerde neuronale kweken en worden in detail beschreven hier. De procedure voor twee immunocytochemie tegen neurale cel adhesiemolecuul (NCAM) en β1 integrines voorzien. Het genereren van deze immunocytochemische werkwijzen zijn gebruikt om de ruimtelijke patronen en intensiteit van verscheidene moleculen in gekweekte neuronen 8,16 onderzoeken.

<table border="0" cellpadding="0" cellspacing="0" width="694">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sterile small culture dishes (35mm)</td>
			<td style="width:176px;">Corning</td>
			<td style="width:135px;">430165</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sterile large culture dishes (100mm)</td>
			<td style="width:176px;">Falcon</td>
			<td style="width:135px;">353003</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sterile large petri dishes (100mm)</td>
			<td style="width:176px;">VWR</td>
			<td style="width:135px;">89000-302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">glass petri dish (100mm)</td>
			<td style="width:176px;">VWR</td>
			<td style="width:135px;">D108962&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">fine forceps</td>
			<td style="width:176px;">Fine Science Tools</td>
			<td style="width:135px;">11251-10&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">standard forceps</td>
			<td style="width:176px;">Fine Science Tools</td>
			<td style="width:135px;">1100-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">coverslips&#160; (22x22)</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:135px;">12 518 105K</td>
			<td>0.13-0.17 mm thickness is optimal for microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">porcelin coverslip holder</td>
			<td style="width:176px;">Thomas scientific</td>
			<td style="width:135px;">8542E40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine serum</td>
			<td style="width:176px;">Gibco</td>
			<td style="width:135px;">17502-048</td>
			<td>use 50ml aliquots per 500 mls of Ham&#39;s F12 media&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HCL</td>
			<td style="width:176px;">VWR</td>
			<td style="width:135px;">VW3204-1</td>
			<td>use at 2M, can be used twice before discarding</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">S9888</td>
			<td>use 5M NaCl solution for laminin-1 stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">laminin-1</td>
			<td style="width:176px;">Invitrogen</td>
			<td style="width:135px;">23017-015</td>
			<td>Add 72ul of sterile 5M NaCl and aliquot into 50ul</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml conical tube</td>
			<td style="width:176px;">cell treat</td>
			<td style="width:135px;">229411</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS CMF 10X</td>
			<td style="width:176px;">Gibco</td>
			<td style="width:135px;">14200</td>
			<td>used at 1X, dilute with sterile millipore filtered water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS 10X</td>
			<td style="width:176px;">Gibco</td>
			<td style="width:135px;">14080</td>
			<td>used at 1X, dilute with sterile milliporee filtered water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ham&#39;s F12</td>
			<td style="width:176px;">Lonza</td>
			<td style="width:135px;">12-615F</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fetal Bovine Serum Albumin (BSA)</td>
			<td style="width:176px;">Gibco</td>
			<td style="width:135px;">10438</td>
			<td>use in F12HS20 at 10%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">H3375</td>
			<td>make sterile 1M solution in distilled water, dilute in F12 media to obtain final concentration of 10mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin streptomyocin</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">P0781</td>
			<td>use 5mls in 500mls of F12 media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NT3</td>
			<td style="width:176px;">Millipore</td>
			<td style="width:135px;">GF031</td>
			<td>Add 1ml of sterile millipore water, aliquot under sterile conditions, store at -20 0C, use at final concentration of 10ng/ml in F12H media, keep in -20 C&#160; non-defrosting freezer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N2</td>
			<td style="width:176px;">Invitrogen</td>
			<td style="width:135px;">17502048</td>
			<td>&#160;aliquot under sterile conditions,stored at -20 0C,&#160; use 100ul per 10ml of F12H media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NGF</td>
			<td style="width:176px;">RnD systems</td>
			<td style="width:135px;">256-GF</td>
			<td>Add 1ml of sterile 1X PBS with 0.1% BSA, aliquot under sterile conditions, store at -20 0C, usd at final concentration of 10 ng/ml in F12H media&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">l-glutamine</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">G7513</td>
			<td>aliquot under sterile conditions, store at -20 0C, use at final concentration of 200mM in F12H media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">T4049</td>
			<td>aliquot under sterile conditions, store at -20 0C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:176px;">Gibco</td>
			<td style="width:135px;">15260</td>
			<td></td>
		</tr>
		<tr height="231">
			<td height="231" style="height:231px;width:216px;">Other items needed:&#160; general dissection instruments, including&#160; glass pasteur pipettes, fertile white leghorn chicken eggs, check egg incubator (humidified, 37 degrees C), cell incubation chamber (humidified, 37&#160; degrees C), laminar flow hood, binocular stereovision dissecting scope</td>
			<td style="width:176px;"></td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Immunocytochemistry Reagents Table</td>
			<td style="width:176px;"></td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sucrose</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">S9378</td>
			<td>used in fixative solution (4% paraformaldehye, 30% sucrose, 2X PBS)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton-X 100</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">T-8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraformaldehyde</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">P6148</td>
			<td>used in fixative solution (4% paraformaldehye, 30% sucrose, 2X PBS)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluoromount G</td>
			<td style="width:176px;">Southern Biotech</td>
			<td style="width:135px;">0100-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">normal goat serum</td>
			<td style="width:176px;">Life technologies</td>
			<td style="width:135px;">PCN500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">microscope slides</td>
			<td style="width:176px;">VWR</td>
			<td style="width:135px;">16004-430</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Primary Antibody Table</td>
			<td style="width:176px;"></td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antibody against NCAM</td>
			<td style="width:176px;">Millipore</td>
			<td style="width:135px;">AB5032</td>
			<td>polyclonal</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Antibody against activated beta 1 ingegrin</td>
			<td style="width:176px;">Millipore</td>
			<td style="width:135px;">MAB19294</td>
			<td>monoclonal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Seconday Antibody Table</td>
			<td style="width:176px;"></td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Goat anti-mouse IgG Alexa 488</td>
			<td style="width:176px;">Life technologies</td>
			<td style="width:135px;">A11001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Goat anti-rabbit IgG Alexa 548</td>
			<td style="width:176px;">Life technologies</td>
			<td style="width:135px;">A11036</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culture of dissociated sensory neurons from chick embryos

Neuronen zijn veelzijdige cellen die informatie essentieel is voor een verscheidenheid aan functies, waaronder sensatie, motoriek, leren en geheugen uit te voeren. Studeren neuronen in vivo kan lastig zijn vanwege hun complexiteit, hun gevarieerde en dynamische omgevingen, en technische beperkingen. Daarom bestuderen neuronen in vitro gunstig kan blijken om de complexe geheimen van neuronen ontrafelen. De goed gedefinieerde aard van celcultuur modellen biedt gedetailleerde controle over de milieu-omstandigheden en variabelen. Hier beschrijven we hoe te isoleren, distantiëren, en cultuur primaire neuronen van kippenembryo&#39;s. Deze techniek is snel, goedkoop en genereert stevig groeiende sensorische neuronen. De procedure produceert vaste culturen die sterk verrijkt voor neuronen en heeft zeer weinig niet-neuronale cellen (minder dan 5%). Primaire neuronen niet goed aan onbehandelde glas of weefselkweek plastic derhalve gedetailleerde procedures twee Distin creërenct, goed gedefinieerde-laminin bevatten substrata voor neuronale plating worden beschreven. Gekweekte neuronen zijn zeer vatbaar voor meerdere cellulaire en moleculaire technieken, met inbegrip van co-immunoprecipitatie, live cell verbeelden, RNAi, en immunocytochemie. Procedures voor dubbel immunocytochemie volgende gekweekte neuronen werden geoptimaliseerd en beschreven.

De hier beschreven protocol stelt onderzoekers cultuur een verrijkte populatie van gedissocieerde embryonale sensorische neuronen met zeer weinig (bijvoorbeeld &lt;5%) van niet-neuronale cellen 7,8,10,16. Tal van DRG&#39;s kan worden verkregen bij de lumbosacrale, thoracale en cervicale regio. Afhankelijk van de behoeften van de onderzoeker, kunnen DRG&#39;s van deze verschillende anatomische gebieden gemakkelijk worden geïsoleerd. Bijvoorbeeld, Figuur 1 toont beelden van het kippen...

Watch this Scientific Journal Video about Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos at JoVE.com

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Celkweekmodellen gedetailleerde controle milieuomstandigheden en dus een krachtig platform om verschillende aspecten van neuronale cel biologie. We beschrijven een snelle, goedkope en betrouwbare wijze te isoleren, dissociëren en cultuur sensorische neuronen van kippenembryo&#39;s. Details van de ondergrond voorbereiding en immuuncytochemie zijn ook aanwezig.

Isolatie en Cultuur van Gedissocieerde sensorische neuronen van Chick Embryo

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Research

JoVE Journal

Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Dissectie en Cultuur van Commissural neuronen van het ruggenmerg Embryonic

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

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Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

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Live-beeldvorming van PKC Translocatie in Sf9 cellen en in Aplysia sensorische neuronen

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Isolatie en Cultuur van hippocampale neuronen van Prenatal Muizen

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

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

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Isolatie van sensorische neuronen van<em&gt; Aplysia californica</em&gt; Voor Patch Clamp Recordings van Glutamaterge Currents

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Productie en isolatie van axonen van sensorische neuronen voor Biochemische analyse met behulp van poreuze filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Isolatie, Cultuur en onderhoud op lange termijn van de Primary mesencefale dopaminerge neuronen uit embryonale knaagdieren Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Stripe Assay aan de Aantrekkelijke of Repulsive activiteit van een eiwit ondergrond met Gescheiden hippocampus Neuronen Studie

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Verwijdering van<em&gt; Drosophila</em&gt; Spier Weefsel van Larvale Filets voor Immunofluorescentie Analyse van sensorische neuronen en opperhuidcellen

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

zebravis<em&gt; In Situ</em&gt; Spinal Cord Voorbereiding voor elektrofysiologische registraties van Spinal sensorische en motorische neuronen

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...