Name: analysis of trunk neural crest cell migration using a modified zigmond chamber assay
Uploaded: 2012-01-19T12:00:00
Description: Watch this Scientific Journal Video about Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay at JoVE.com

We give special thanks to Lino Kim, Steve Guzman and Ujit Satyarthi for technical assistance during the development of this method. Myron Hawthorne, Richard Spengel, and Roberto Rojas machined the chambers used here and provided much-needed technical assistance. Notably, Roberto Rojas produced Figure 4. We are also thankful for Scott Fraser&#x2019;s invaluable advice prior to the development of the above chemotaxis assay. This work was partly supported by an NIH-MBRS SCORE-5S06GM048680-13 to MEdB and by an award from the CSU, Northridge Graduate Thesis Support Program to CW.

1. Jour 1: Isolement des tubes du tronc de neurones pour une nuit de culture sur des lamelles <ol><li> Oeufs chiches Incuber 56 h à 38 ° C. Retirer les oeufs de l&#39;incubation, les pulvériser légèrement à l&#39;éthanol 70%, puis laissez-les sécher. Casser les oeufs s&#39;ouvrent sur un plateau en verre stérilisée aux UV. </li><li> Extrait chaque embryon de son jaune d&#39;oeuf et le placer dans chiches Ringer. Pour ce faire, la première coupe autour de ses îles de sang avec des ciseaux courbes, puis, ave...

<ol>
	<li>Le Douarin, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+avian+embryo+as+a+model+to+study+the+development+of+the+neural+crest:+a+long+and+still+ongoing+story.">The avian embryo as a model to study the development of the neural crest: a long and still ongoing story.</a> Mechanisms of Development. 121, 1089-1102 (2004).</li><li>Baker, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neural Crest and Cranial Ectodermal Placodes. , (2005).</li><li>Gammill, L. S., Roffers-Agarwal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Division+of+labor+during+trunk+neural+crest+development.">Division of labor during trunk neural crest development.</a> Dev. Biol. 344, 555-565 (2010).</li><li>Kulesa, P. M., Gammill, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+migration:+patterns,+phases+and+signals.">Neural crest migration: patterns, phases and signals.</a> Dev. Biol. 344, 566-568 (2010).</li><li>Wang, H. U., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eph+family+transmembrane+ligands+can+mediate+repulsive+guidance+of+trunk+neural+crest+migration+and+motor+axon+outgrowth.">Eph family transmembrane ligands can mediate repulsive guidance of trunk neural crest migration and motor axon outgrowth.</a> Neuron. 18, 383-396 (1997).</li><li>Krull, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+Eph-related+receptors+and+ligands+confer+rostrocaudal+pattern+to+trunk+neural+crest+migration.">Interactions of Eph-related receptors and ligands confer rostrocaudal pattern to trunk neural crest migration.</a> Curr. Biol. 7, 571-580 (1997).</li><li>Gammill, L. S., Gonzalez, C., Gu, C., Bronner-Fraser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidance+of+trunk+neural+crest+migration+requires+neuropilin+2/semaphorin+3F+signaling.">Guidance of trunk neural crest migration requires neuropilin 2/semaphorin 3F signaling.</a> Development, Cambridge, England. , 133-199 (2006).</li><li>De Bellard, M. E., Rao, Y., Bronner-Fraser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+function+of+Slit2+in+repulsion+and+enhanced+migration+of+trunk,+but+not+vagal,+neural+crest+cells.">Dual function of Slit2 in repulsion and enhanced migration of trunk, but not vagal, neural crest cells.</a> The Journal of cell biology. 162, 269-279 (2003).</li><li>Kasemeier-Kulesa, J. C., McLennan, R., Romine, M. H., Kulesa, P. M., Lefcort, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCR4+controls+ventral+migration+of+sympathetic+precursor+cells.">CXCR4 controls ventral migration of sympathetic precursor cells.</a> J. Neurosci. 30, 13078-13088 (2010).</li><li>Zigmond, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ability+of+polymorphonuclear+leukocytes+to+orient+in+gradients+of+chemotactic+factors.">Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors.</a> The Journal of Cell Biology. 75, 606-616 (1977).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chicken+embryo.">A series of normal stages in the development of the chicken embryo.</a> J. Morph. 88, 49-52 (1951).</li><li>Boyden, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemotactic+effect+of+mixtures+of+antibody+and+antigen+on+polymorphonuclear+leucocytes.">The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes.</a> The Journal of Experimental Medicine. 115, 453-466 (1962).</li><li>Davis, E. M., Trinkaus, 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=Significance+of+cell-to+cell+contacts+for+the+directional+movement+of+neural+crest+cells+within+a+hydrated+collagen+lattice.">Significance of cell-to cell contacts for the directional movement of neural crest cells within a hydrated collagen lattice.</a> Journal of Embryology and Experimental Morphology. 63, 29-51 (1981).</li><li>Zicha, D., Dunn, G. A., Brown, A. 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Conducting chemotaxis research on trunk NCCs has proven challenging for a series of reasons. Trunk NCCs constitute a heterogeneous stem cell population that will differentiate if cultured long-term; therefore, trunk NCCs must be obtained from primary explantation of the trunk-level NT. Conventional methods to study the chemotactic response of homogenously distributed cell populations in vitro are difficult to test on trunk NCCs since they first require that cells are isolated and homogenously replated in ...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Omega Scientific</td>
			<td>DM-22</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin Solution</td>
			<td>Omega Scientific</td>
			<td>PS-20</td>
			<td>100X Stock Concentration</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Omega Scientific</td>
			<td>GS-60</td>
			<td>100X Stock Concentration</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Omega Scientific</td>
			<td>FB-11</td>
			<td>Lot# 105247 (or another that is comparable)</td>
		</tr>
		<tr>
			<td>Modified Zigmond chamber</td>
			<td>Home made</td>
			<td>N/A</td>
			<td>Reservoir volume: ~ 160 &mu;l ea; for additional specifications, see Fig. 4 and the supplemental fabrication protocol</td>
		</tr>
		<tr>
			<td>Cell culture dish</td>
			<td>Denville</td>
			<td>T6040</td>
			<td>40 x 10 mm</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>BD</td>
			<td>354008</td>
			<td>10X Stock prepped by diluting 1 mg FN in 1 ml H2O and 9 ml DMEM</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Fisher</td>
			<td>12-548-B</td>
			<td>Precleaned; 22 x 22 mm</td>
		</tr>
		<tr>
			<td>L15 medium</td>
			<td>Thermo Scientific</td>
			<td>SH30525.02</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Petroleum Jelly</td>
			<td>Comforts</td>
			<td>011110794642</td>
			<td>100%</td>
		</tr>
		<tr>
			<td>Centrifuge tube</td>
			<td>Biologix</td>
			<td>10-9152</td>
			<td>15 ml</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Cell Systems</td>
			<td>4Z0-850</td>
			<td>10X Stock Concentration</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD</td>
			<td>309602</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>Needle</td>
			<td>BD</td>
			<td>305127</td>
			<td>25 G x 1.5 in.</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-IgM</td>
			<td>Invitrogen</td>
			<td>A21042</td>
			<td>Stock is 2 mg/ml; 7 moles dye/mole IgM</td>
		</tr>
		<tr>
			<td>Dissecting Forceps</td>
			<td>FST</td>
			<td>Misc.</td>
			<td>Dumont #5 or 55; straight tipped; stainless steel or titanium</td>
		</tr>
		<tr>
			<td>Tungsten Needle</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Home made; placed in a pin holder</td>
		</tr>
		<tr>
			<td>Blunt Forceps</td>
			<td>Tiemann</td>
			<td>160-18</td>
			<td>Used for transferring embryos to Ringer's from egg yolk</td>
		</tr>
	</tbody>
</table>
Supplemental Protocol: Fabrication of a Modified Zigmond Chamber
Please refer to Figure 4 as a reference for the protocol below:
<ol>
	<li>Purchase a sheet of 3/16" thick polished acrylic (4.45 mm actual thickness).</li>
	<li>Using a table saw, cut chamber blanks oversized to the rough dimensions of 33.25 mm x 64.57 mm. This allows 3.175 mm extra material for machining.</li>
	<li>Set the chamber blank on a vise. With a milling machine and a 6.35 mm (1/4") end mill bit, finish machining the sides of the chamber to their exact dimensions: 30.07 mm x 61.39 mm.</li>
	<li>Position the chamber blank on the milling machine and locate the center of the blank along both the x and y axes with an edge finder; then zero the center location.</li>
	<li>Acquire the chamber height (z-axis) by touching the end mill bit to the top surface and zero the height.</li>
	<li>Using a 3.91 mm (0.154") end mill bit, offset the bit 3.03 mm along the x-axis (positive direction) for the first reservoir. Begin machining into the chamber to a depth of 2.84 mm while moving along the y-axis (positive direction) to 7.62 mm (0.300") and then traverse to 7.62 mm (0.300") in the opposite (negative) direction to a complete reservoir length of 15.24 mm (0.600"). Offset the bit to 3.03 mm (0.119") along the x-axis (negative direction) and repeat the same process for the second reservoir.</li>
	<li>Position the chamber on its edge and drill a hole using a 1.09 mm (0.043 in.) drill bit on the end of each reservoir (4 total) that connects the end of the reservoir to the side of the chamber for loading medium during experimentation.</li>
	<li>Soak the chamber well in warm soapy water to help remove any chemical contaminants.</li>
	<li>Soak and rinse the chamber well in double-distilled water to remove any soap. The chambers are now ready to use as described above.</li>
</ol>

analysis of trunk neural crest cell migration using a modified zigmond chamber assay

Cellules de la crête neurale (CCN) sont une population de passage de cellules présentes dans le développement des vertébrés qui émigrent à partir du tube neural dorsal (NT) après avoir subi une 1,2 transition épithélio-mésenchymateuses. Après EMT, CNC migrer de grandes distances le long des voies stéréotypés jusqu&#39;à ce qu&#39;ils atteignent leurs objectifs. CNC se différencier en un vaste éventail de types de cellules, y compris les neurones, cellules gliales, les mélanocytes et les cellules chromaffines 1-3. La capacité du CNC à atteindre et à reconnaître leurs emplacements cible appropriée est fondamentale pour la formation appropriée de toutes les structures contenant le tronc de la CCN composants dérivés 3. Élucider les mécanismes d&#39;orientation pour le tronc de la CCN de migration a donc été un sujet de grande importance. Plusieurs molécules ont été démontrés pour guider la CCN migrations 4. Par exemple, les CNC du tronc sont connus pour être repoussés par des signaux de guidage négatifs tels que Semaphorin, Ephrine et les ligands fente 5-8. Cependant, ne pasjusqu&#39;à récemment toute chimiotactiques du CNC du tronc été identifiés 9. Conventionnel dans les approches in vitro pour étudier le comportement chimiotactique de cellules adhérentes mieux travailler avec immortalisé, de façon homogène les cellules distribuées, mais sont plus difficiles à appliquer à certaines cultures primaires de cellules souches qui n&#39;ont pas d&#39;abord une distribution homogène et rapide de différencier (comme CNC). Une approche pour homogénéiser la répartition de la CNE du tronc pour les études de chimiotactisme est d&#39;isoler les CNC du tronc de l&#39;enseignement primaire des cultures d&#39;explants NT, puis soulevez et replate qu&#39;ils soient près de 100% de confluence. Cependant, cette approche nécessite plaquage des quantités substantielles de temps et d&#39;effort pour explant suffisamment de cellules, est rude, et distribue des CCN du tronc d&#39;une manière différente à celle trouvée dans des conditions in vivo. Nous rapportons ici une approche in vitro, qui est en mesure d&#39;évaluer la chimiotaxie et d&#39;autres réponses migratoires des CNC tronc sans requirindistribution de ga cellulaire homogène. Cette technique utilise imagerie time-lapse du primaire, imperturbable CNC tronc intérieur d&#39;une chambre modifiés Zigmond (une chambre standard de Zigmond est décrit ailleurs 10). En exposant CNC tronc à la périphérie de la culture à un gradient de chemotactant qui est perpendiculaire à leur directivité naturelle prédit, des altérations de la polarité migratoires induits par le gradient chemotactant appliquée peut être détecté. Cette technique est peu coûteuse, nécessite la mise en culture d&#39;explants NT seulement deux pour le traitement de reproduire, de levage évite de cellules dures (telles que la trypsine), laisse les CNC malle dans une distribution plus semblable à des conditions in vivo, réduit la quantité de temps entre l&#39;explantation et l&#39;expérimentation (ce qui réduit probablement le risque de différenciation), et permet time-lapse d&#39;évaluation des caractéristiques de nombreux migrateurs.

Watch this Scientific Journal Video about Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay at JoVE.com

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

Analyse de la migration cellulaire Tronc Crête neurale en utilisant une modification Zigmond Chambre Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

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