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

Vi vil gjerne takke Alison Philbrook, Belinda Barbagallo og Michael Francis for innsiktsfulle kommentarer på dette papiret. Forskning rapportert i denne publikasjonen ble støttet av en R15 AREA award 1R15NS070172-01A1 tildelt MLL.

1. Cover Forberedelse: Acid Wash og Bake <ol><li> Minst to dager før disseksjon (trinn 2), begynner de følgende skritt for å forberede Dekk. Utfør syre vasketrinn for å fjerne oljer og grundig rene dekkglass. </li><li> Belastningsdekk i porselen holder som vertikalt posisjonerer Dekk og hindrer dem fra å berøre hverandre. Senk holderen og dekkglass i glass histologi beholder fylt med 2 M saltsyre (HCl). Alternativt, hvis porselen innehaveren ikke er tilgjengelig, spre ~ 20 Dekk horisontalt for å dekke bunnen p...

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Her presenterer vi detaljerte protokoller for isolering og dyrking dissociated sensoriske nerveceller fra en kylling embryo. Denne prosedyren genererer en beriket befolkning på robust voksende nevroner in vitro 7,8,10,16. Mange cellulære og molekylære teknikker kan anvendes på disse dyrkede nevroner, inkludert immunocytokjemi, som er beskrevet her. Denne protokollen ble nylig brukt til kvantitativt vurdere intensiteten av immunolabeled aktivert inte i sensoriske vekst kjegler 8,16. I d...

Neuroner er komplekse celler som bærer informasjon som er viktig for en rekke funksjoner, inkludert følelse, syn, motorbevegelse, læring og hukommelse. Unike fra andre celletyper, neuroner strekker arm-lignende prosesser, kalt axoner, for å danne nødvendige nevrale motorveier for kommunikasjon. Under utvikling spesialisert avdelinger plassert på tuppen av voksende axoner, kalt vekst kjegler, navigere gjennom en konsert av ekstracellulære signaler for å lede axon til dens passende destinasjon. Den intrikate molekylære mekanismene som ligger bak veksten kjegle navigasjon er ikke fullt ut forstått. For bedre å forstå disse mekanismene, har etterforskerne brukt cellekultur modeller for å studere nervecellene i et definert og forenklet in vitro miljø. Studerer nevroner i kultur en har ført til betydelige fremskritt i vår forståelse av nervecellebiologi inkludert: neuronal differensiering 2, cytoskeletal dynamikk, endocytose og trafficking, Dendrittregulering 3,4, aksonal regenerasjon 5, og kliniske tilstander som neuropathies seks. I tillegg, dyrkede neuroner er svært mottagelig for et bredt spekter av forskning teknikker inkludert immunocytokjemi, celleoverflate co-immunoutfelling, Western blot, transfeksjon, RNAi, og direkte avbildning slik som timelapse analyse av vekst kjegle motilitet. Således dyrkning primære neuroner er en kraftig metode for å belyse en rekke sider ved cellebiologi av neuroner. Cellekulturmodell gir etterforskere med detaljert kontroll over miljøforhold og variabler. For eksempel kan substratet på hvilke neuroner er belagt (og vokse over) lett manipuleres. Her vi gi detaljerte instruksjoner for å generere to forskjellige substrater, en med lav laminin-1-konsentrasjon, og den andre med mettende konsentrasjoner av laminin-1. Overraskende, kan forskjellige konsentrasjoner av det samme molekylet har dramatiske effekterpå den interne tilstand av nerveceller, så vel som deres celleoverflate sammensetning. For eksempel, intracellulære nivåer av cAMP og overflate nivåer av inte er vesentlig forskjellige i nevroner belagt på disse to grunnen 7,8. Ytterligere studier har vist at andre molekyler, inkludert fibronektin og chondroitin sulfat proteoglykaner, påvirker ekspresjonen av celleoverflatemolekyler og neuronal motilitet 7-11. I tillegg er løselige molekyler som neurotrophins og neurotropins også påvirke cellemembranen sammensetning og neuronal motilitet 12-16 og kan lett og nøyaktig manipuleres i en cellekulturmodell. Her beskriver vi metoder for å isolere og kultur dissociated sensoriske nerveceller fra kyllingembryo. Denne prosedyren har blitt brukt til å gjøre betydelige gjennombrudd i nevrobiologi, inkludert axon utvekst 5,7,8,10,11,16-21 og ble endret fra en prosedyre utviklet for å isolere ganglion celler 22. Det finnesflere fordeler med denne tilnærmingen. Først er mange funksjoner i chick rygg rotganglion (DRG) utvikling godt karakterisert inkludert tidsramme for fødselen, axon forlengelse, og protein uttrykk profiler 2,23-28, og dermed gi en instruktiv grunnlaget for å bygge informativ in vitro eksperimenter. For det andre, dissosiert neuronale kulturer tillate undersøkeren til mer direkte å studere neuroner sammenlignet med alternative metoder ved bruk av intakte DRG-eksplantater (som inneholder neuroner og ikke-neuronale celler) og / eller blandede kulturer inneholdende både dissosiert nevroner og ikke-nevronale celler. Tredje, den prosedyren som er beskrevet her er enkel, billig og mottagelig for studenter. Derfor kan denne teknikken brukes til forskning, så vel som for undervisningsformål. Videre bør mindre variasjoner av denne protokollen tillater rask, høy avkastning rensing av nerveceller fra andre enn DRGs kilder. For eksempel kan denne prosedyren bli endret for å gi neuronally enriched kulturer fra andre vev slik som embryonisk forhjerne eller ryggmargen. Immunocytochemistry protokoller har blitt optimalisert for disse dissociated nevrale kulturer og er beskrevet i detalj her. Fremgangsmåten for dobbelt immunocytokjemi mot neural celleadhesjonsmolekyl (NCAM) og β1 integriner er gitt. Data som genereres fra disse immuncytokjemisk metoder har blitt brukt til å undersøke den romlige mønster og intensitet av flere molekyler i dyrkede nerveceller 8,16.

<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

Nerveceller er mangefasetterte celler som bærer informasjon avgjørende for en rekke funksjoner, inkludert følelse, motorikk, læring og hukommelse. Studerer nevroner in vivo kan være utfordrende på grunn av sin kompleksitet, sine varierte og dynamiske miljøer, og tekniske begrensninger. Av disse grunner, kan studere nerveceller in vitro være gunstig å løse de komplekse mysterier nevroner. Den godt definerte art cellekulturmodeller gir detaljert kontroll med miljøforhold og variabler. Her beskriver vi hvordan du isolerer, distansere, og kultur primære nerveceller fra kyllingembryo. Denne teknikken er rask, billig, og genererer robustly voksende sensoriske nerveceller. Fremgangsmåten produserer konsekvent kulturer som er sterkt anriket på neuroner og har svært få ikke-neuronale celler (mindre enn 5%). Primære nevroner ikke holder seg godt til ubehandlet glass eller vev kultur plast, derfor detaljerte prosedyrer for å lage to Distinct, er veldefinert laminin-inneholdende substratet for neuronal plette beskrevet. Dyrkede nevroner er svært mottagelig for flere cellulære og molekylære teknikker, inkludert co-immunoprecipitation, levende celle innbiller seg, RNAi, og immunocytochemistry. Prosedyrer for dobbel immunocytochemistry på følgende dyrkede nerveceller har blitt optimalisert og beskrevet her.

Protokollen er beskrevet her gjør etterforskere til kultur en beriket befolkning på dissociated embryonale sensoriske nevroner med svært få (f.eks &lt;5%) ikke-nevronale celler 7,8,10,16. Mange DRGs kan fås fra lumbosacral, thorax og cervical regioner. Avhengig av behovene til etterforsker, kan DRGs fra disse forskjellige anatomiske regioner være lett isolert. For eksempel, Figur 1 viser bilder av kylling embryo gjennom ulike stadier av disseksjon med lende DRGs uthevet i <stro...

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

Cellekultur modellene gir detaljert kontroll over miljøforhold og dermed gi en kraftig plattform for å belyse mange aspekter av nevronale cellebiologi. Vi beskriver en hurtig, billig og pålitelig metode for å isolere, dissosierer, og kultur sensoriske neuroner fra kyllingembryo. Detaljer om grunnen forberedelse og immunocytochemistry tilbys også.

Isolasjon og kultur av Spaltet sensoriske nevroner Fra Chick embryo

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Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

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 ...

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Isolering og kultur hippocampus Nerveceller fra Prenatal 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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Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

Isolering av sensoriske nevroner av<em&gt; Aplysia californica</em&gt; For Patch Clamp Recordings i glutamaterg 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

Produksjon og Isolering av Axoner fra sensoriske nevroner for Biokjemisk analyse ved hjelp Porøse 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

Isolasjon, Kultur og langsiktig vedlikehold av Primary mesencefalisk dopaminerge nevroner Fra Embryonic Gnagere 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-analyse for å studere attraktiv eller frastøtende aktivitet av et protein underlaget med Spaltet hippocampus nevroner

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

fjerning av<em&gt; Drosophila</em&gt; Muskelvev fra Larve Fileter for Immunofluorescensanalyse av sensoriske nevroner og epidermal cellene

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

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