We thank Corinne A. Fairchild and Katie L. Vermillion, who participated in developing our version of the chick cranial neural fold culture protocol.

Any variety of Gallus gallus breeds may be used, including White Leghorn, Golden Sex Link, or Rhode Island Red. The chicken eggs used in the present study were of various breeds and obtained from multiple sources, including local farms and hatcheries.
1. Preparation of solutions and materials
<ol>
	<li>Prepare Ringer&#39;s solution by mixing 123.3 mM NaCl, 1.53 mM CaCl2, 4.96 mM KCl, 0.809 mM Na2HPO4, and 0.147 mM KH2PO<su...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-to-mesenchymal+transition+and+different+migration+strategies+as+viewed+from+the+neural+crest.">Epithelial-to-mesenchymal transition and different migration strategies as viewed from the neural crest.</a> Current Opinion in Cell Biology. 66, 43-50 (2020).</li><li>McLennan, R., 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=Neural+crest+cells+bulldoze+through+the+microenvironment+using+Aquaporin+1+to+stabilize+filopodia.">Neural crest cells bulldoze through the microenvironment using Aquaporin 1 to stabilize filopodia.</a> Development. 147 (1), 185231 (2020).</li><li>Carmona-Fontaine, C., 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=Complement+fragment+C3a+controls+mutual+cell+attraction+during+collective+cell+migration.">Complement fragment C3a controls mutual cell attraction during collective cell migration.</a> Developmental Cell. 21 (6), 1026-1037 (2011).</li><li>Giovannone, D., 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=Slits+affect+the+timely+migration+of+neural+crest+cells+via+robo+receptor.">Slits affect the timely migration of neural crest cells via robo receptor.</a> Developmental Dynamics. 241 (8), 1274-1288 (2012).</li><li>Vermillion, K. L., Lidberg, K. A., 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=Cytoplasmic+protein+methylation+is+essential+for+neural+crest+migration.">Cytoplasmic protein methylation is essential for neural crest migration.</a> Journal of Cell Biology. 204 (1), 95-109 (2014).</li><li>Yang, X., Li, J., Zeng, W., Li, C., Mao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elongator+Protein+3+(Elp3)+stabilizes+Snail1+and+regulates+neural+crest+migration+in+Xenopus.">Elongator Protein 3 (Elp3) stabilizes Snail1 and regulates neural crest migration in Xenopus.</a> Scientific Reports. 6 (1), 1-9 (2016).</li><li>Gonzalez Malagon, S. G., 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=Glycogen+synthase+kinase+3+controls+migration+of+the+neural+crest+lineage+in+mouse+and+Xenopus.">Glycogen synthase kinase 3 controls migration of the neural crest lineage in mouse and Xenopus.</a> Nature Communications. 9 (1), 1-15 (2018).</li><li>Bhattacharya, D., Azambuja, A. P., Simoes-Costa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+reprogramming+promotes+neural+crest+migration+via+yap/tead+signaling.">Metabolic reprogramming promotes neural crest migration via yap/tead signaling.</a> Developmental Cell. 53 (2), 199-211 (2020).</li><li>Jacques-Fricke, B. T., 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=Profiling+NSD3-dependent+neural+crest+gene+expression+reveals+known+and+novel+candidate+regulatory+factors.">Profiling NSD3-dependent neural crest gene expression reveals known and novel candidate regulatory factors.</a> Developmental Biology. 475, 118-130 (2021).</li><li>Bronner-Fraser, M., Garc&#237;a-Castro, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+4+manipulations+of+neural+crest+cells+or+their+migratory+pathways.">Chapter 4 manipulations of neural crest cells or their migratory pathways.</a> Methods in Cell Biology. 87, 75-96 (2008).</li><li>Milet, C., Monsoro-Burq, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+xenopus+laevis+neural+crest+for+in+vitro+explant+culture+or+in+vivo+transplantation.">Dissection of xenopus laevis neural crest for in vitro explant culture or in vivo transplantation.</a> Journal of Visualized Experiments. (85), e51118 (2014).</li><li>Malagon, S. G. G., 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=Dissection,+culture+and+analysis+of+primary+cranial+neural+crest+cells+from+mouse+for+the+study+of+neural+crest+cell+delamination+and+migration.">Dissection, culture and analysis of primary cranial neural crest cells from mouse for the study of neural crest cell delamination and migration.</a> Journal of Visualized Experiments. (152), e60051 (2019).</li><li>Theveneau, E., Mayor, R. <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+delamination+and+migration:+From+epithelium-to-mesenchyme+transition+to+collective+cell+migration.">Neural crest delamination and migration: From epithelium-to-mesenchyme transition to collective cell migration.</a> Developmental Biology. 366 (1), 34-54 (2012).</li><li>Conrad, G. W., Bee, J. A., Roche, S. M., Teillet, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+microscalpels+by+electrolysis+of+tungsten+wire+in+a+meniscus.">Fabrication of microscalpels by electrolysis of tungsten wire in a meniscus.</a> Journal of Neuroscience Methods. 50 (1), 123-127 (1993).</li><li>Hamburger, V., Hamilton, H. 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S., Jacques-Fricke, B., 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=Embryological+and+genetic+manipulation+of+chick+development.">Embryological and genetic manipulation of chick development.</a> Methods in Molecular Biology. 1920, 75-97 (2019).</li><li>Vandekerckhove, J., Deboben, A., Nassal, M., Wieland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phalloidin+binding+site+of+F-actin.">The phalloidin binding site of F-actin.</a> The EMBO Journal. 4 (11), 2815-2818 (1985).</li><li>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=Analysis+of+the+early+stages+of+trunk+neural+crest+migration+in+avian+embryos+using+monoclonal+antibody+HNK-1.">Analysis of the early stages of trunk neural crest migration in avian embryos using monoclonal antibody HNK-1.</a> Developmental Biology. 115 (1), 44-55 (1986).</li><li>Schindelin, 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=Fiji:+An+open-source+platform+for+biological-image+analysis.">Fiji: An open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Soille, P., Vincent, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+watersheds+in+digital+pictures+via+flooding+simulations.">Determining watersheds in digital pictures via flooding simulations.</a> Visual Communications and Image Processing '90: Fifth in a Series. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+ovo+live+imaging+of+avian+embryos.">In ovo live imaging of avian embryos.</a> Cold Spring Harbor Protocols. 5 (6), (2010).</li><li>McKinney, M. C., Kulesa, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+the+neural+crest+cell+epithelial-to-mesenchymal+transition+in+the+chick+embryo.">Live imaging of the neural crest cell epithelial-to-mesenchymal transition in the chick embryo.</a> Methods in Molecular Biology. 2179, 107-114 (2021).</li><li>Gustafson, C. M., Roffers-Agarwal, J., Gammill, L. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+morpholinos+to+control+gene+expression.">Using morpholinos to control gene expression.</a> Current Protocols in Nucleic Acid Chemistry. 68 (1), 4-30 (2017).</li><li>Gandhi, S., 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=A+single-plasmid+approach+for+genome+editing+coupled+with+long-term+lineage+analysis+in+chick+embryos.">A single-plasmid approach for genome editing coupled with long-term lineage analysis in chick embryos.</a> Development. 148 (7), (2021).</li><li>Williams, R. 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The authors have no conflicts of interest.

The technique described here provides an adaptable method of isolating chick neural folds and plating them to create cultures of migratory cranial NCCs. These cultures provide simplified 2D conditions for easy analysis of chick NCC migration and morphology that can supplement more technically challenging in ovo imaging methods24,25,26. While this in vitro method is relatively simple, consistent results depend o...

Neural crest cells (NCCs) are a transient cell population in vertebrate embryos. NCCs are specified at the borders of the neural plate and undergo an epithelial-to-mesenchymal transition (EMT) to migrate from the dorsal neural tube1. After EMT, NCCs disperse extensively throughout the embryo, ultimately differentiating and contributing to various structures, including the craniofacial skeleton, outflow tract of the heart, and the majority of the peripheral nervous system2. Changes in cell polarity, the cytoskeleton, and adhesion properties underly this shift from a premigratory to a migratory cell population3. Studying NCC EMT and migration provides insights into fundamental mechanisms of cell motility and informs efforts to prevent and treat birth defects and cancer metastasis.
While in vivo analysis is vital for understanding NCC developmental processes in an embryonic context, in vitro methods offer visual and physical accessibility that facilitate additional experimental avenues. In a simplified 2D environment, NCC morphology, cytoskeletal structures, and distance migrated can be evaluated. Moreover, the effects of genetic or soluble factor perturbation on migratory behaviors of motile NCCs can be analyzed4,5,6,7,8,9,10. In addition, isolated premigratory or migratory NCCs can be collected, pooled, and used for high-throughput methodologies to study the developmental regulation of NCCs through proteomic, transcriptomic, and epigenomic profiling7,11. While methods are available for preparing cranial NCCs from various developmental model organisms12,13,14, this article demonstrates the mechanics of the approach for those first learning to culture cranial NCC from chick embryos.
The current protocol describes a versatile technique for preparing chick cranial NCC cultures (Figure 1). Because NCCs migrate readily from explanted neural folds onto a culture substrate, chick NCCs naturally segregate from embryonic tissue, and primary cultures are easily generated. As midbrain NCCs migrate&#160;en masse&#160;from the cranial neural folds (in contrast to the protracted, cell-by-cell delamination in the trunk15), these cultures consist mainly of migratory cranial neural crest cells, with initial neural fold excision providing a collection method for premigratory NCCs.&#160;A basic method for dissecting and culturing chick cranial neural folds is detailed, and suggestions for different applications and variations on this method are offered.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63799/63799fig01v2.jpg" /> 
Figure 1: Schematic overview of the chick cranial neural fold culture protocol. (A,B) Cranial neural folds (outlined in blue) are excised from a chick embryo with five somites (shown in dorsal view in A). Grey bands, cardiac crescent. (C) When plated on fibronectin, migratory neural crest cells emerge from the neural folds and disperse onto the substrate. <a href="https://www.jove.com/files/ftp_upload/63799/63799fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AxioObserver equipped with an LSM710 confocal scan head controlled by ZEN 3.0 SR software&#160;</td>
			<td>Zeiss</td>
			<td></td>
			<td>Used alpha Plan-Apochromat 100x/1.46 Oil DIC M27 objective</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Chamber dishes (glass bottom, single or divided)</td>
			<td>MatTek; Cell Vis</td>
			<td>P35G-1.5-14-C (MatTek) X000NOJQGX (Cellvis) 
			X000NOK1OJ (Cellvis)</td>
			<td>Single chamber 35 mm or 4 chamber 35 mm</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Carolina Biological Supply Company</td>
			<td>633029, 633031, 633033, 633035, 633037</td>
			<td>circles, 0.13&#8211;0.17 mm thickness, available in 12-25 mm diameter&#160;</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>ThermoFisher Scientific</td>
			<td>11320033</td>
			<td>Alternative for L15 media</td>
		</tr>
		<tr>
			<td>Egg incubator</td>
			<td>Sportsman</td>
			<td>1502</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS&#160;</td>
			<td>Life Technologies</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Fisher Scientific</td>
			<td>CB-40008A</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Whatman</td>
			<td></td>
			<td>grade 3MM chromatography</td>
		</tr>
		<tr>
			<td>Forceps (blunt)</td>
			<td>Fisher Scientific; Thomas Scientific</td>
			<td>08-890 (Fisher);1141W97 (Thomas)</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps (fine)</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td>Dumont #5</td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>https://fiji.sc/</td>
			<td></td>
			<td>Free image analysis software</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr>
			<td>L15 media</td>
			<td>Invitrogen</td>
			<td>11415064</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Media (Vectashield or ProLong Gold)</td>
			<td>Vector Laboratories; Thermofisher Scientific</td>
			<td>H-1700 (Vectashield); P36930 (ProLong Gold)</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-148</td>
			<td>10,000 Units/mL Penicillin; 10,000 mg/mL Streptomycin</td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>VWR (or similar)</td>
			<td></td>
			<td>60 mm, 100 mm</td>
		</tr>
		<tr>
			<td>Phalloidin</td>
			<td>Sigma-Aldrich</td>
			<td>P1951</td>
			<td>multiple flurophores available</td>
		</tr>
		<tr>
			<td>Pin holder</td>
			<td>Fine Science Tools</td>
			<td>26016-12</td>
			<td>For tungsten needle (alternative for spring scissors)</td>
		</tr>
		<tr>
			<td>Scissors (dissection)</td>
			<td>Fine Science Tools</td>
			<td>14061-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td>2.5 mm cutting edge (alternative for tungsten needle)</td>
		</tr>
		<tr>
			<td>Sylgard</td>
			<td>Krayden</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filters</td>
			<td>Sigma-Aldrich</td>
			<td>SLGVM33RS</td>
			<td>Millex-GV Syringe Filter Unit, 0.22 &#181;m, PVDF, 33 mm, gamma sterilized</td>
		</tr>
		<tr>
			<td>Tissue culture dishes</td>
			<td>Sarstedt</td>
			<td>83-3900</td>
			<td>35 mm culture dishes for bulk neural fold cultures</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten wire</td>
			<td>Variety of sources</td>
			<td></td>
			<td>0.01&#34; diameter for tungsten needle (alternative for spring scissors)</td>
		</tr>
	</tbody>
</table>

preparation and morphological analysis of chick cranial neural crest cell cultures

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.

An overview of the present protocol is shown in Figure 1. The incubated eggs were opened, and the yolk, with the embryo on the surface, was isolated by gently pouring into the palm of a gloved hand (Figure 2A,B). After clearing away the albumin (Figure 2C), filter paper frames were applied to the yolk membrane surrounding the embryo to facilitate cutting and lifting the embryo from the yolk, which begins to spill aw...

Watch this Scientific Journal Video about Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures at JoVE.com

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

This versatile protocol describes the isolation of premigratory neural crest cells (NCCs) through the excision of cranial neural folds from chick embryos. Upon plating and incubation, migratory NCCs emerge from neural fold explants, allowing for assessment of cell morphology and migration in a simplified 2D environment.

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like ...

Neural crest cells emigrate from cranial neural folds in embryos or when placed in culture. This provides a convenient method to isolate primary migratory neural crest cells without cell dissociation. While neural crest migration typically occurs within the three-dimensional embryonic environment, two-dimensional culture enables visualization and investigation of migration-related processes.Although cranial neural fold culture protocols exist, this method requires training and organism-specific steps. This video demonstrates techniques to facilitate the reproducible preparation of chick cranial neural fold cultures. Beginners often struggle with dissecting and plating neural folds and assessing culture outcomes.Following the dissection guidelines and applying the morphometric analysis described here allows consistent, quantitative results to be obtained. To begin, place the fertilized eggs in an upright position inside a humidified incubator at 38 degrees Celsius. After removing the eggs from the incubator, disinfect the shells by thoroughly spraying 70%ethanol and letting them dry.For fixing and staining the cultures, use glass cover slips placed into a multi-well tissue culture dish, and pipette enough fibronectin solution to cover the bottom of the well. Next, replace the lid, and incubate the plate with fibronectin solution in a humidified incubator at 38 degrees Celsius for at least one hour while dissecting neural folds. Use blunt forceps to poke a small hole in the shell at 1/4 to 1/3 of the egg's length.Carefully insert the blunt forceps into the small hole, ensuring not to disrupt the yolk. Then, cut through the eggshell around the egg, remove the top of the eggshell, and position the embryo for isolation. Prepare the embryo by gently using the flat edge of closed blunt forceps to wipe away any excess albumin on the yolk's surface covering the embryo.Use forceps to place a filter paper support frame over the embryo, with the embryo in the frame's window. Gently press down on the filter paper to adhere to the yolk. Cut around the outside of the filter paper frame with dissection scissors.Use forceps or scissor tips to grasp the edge of the frame, and gently lift the embryo away from the yolk. Place the embryo with the paper frame side down into a 60 or 100-milliliter Petri dish filled with Ringer's with pen-strep. Keep the dish of embryos on ice if collecting them for an RNA or protein-sensitive downstream application.Transfer an embryo to a clean dish containing Ringer's pen-strep solution. Gently swish the embryo back and forth to clear away any yolk that obscures the view, and exchange the Ringer's pen-strep solution or transfer it to a fresh dish if it becomes cloudy. Position the embryo dorsal and frame side up under a dissecting microscope.Leave the embryo on the paper frame to keep it stretched and held in place. Then, remove the vitelline membrane, using forceps to expose the neural folds. Include tissue caudal to the expanding optic vesicles and rostral to the hindbrain, where rhombomere constrictions are just beginning to appear.Using spring scissors, carefully excise the midbrain neural folds. Take care to excise the dorsal-most aspect of the neural fold with minimal contamination of the neural tube and non-neural ectoderm, and transfer the neural folds to a clean dish containing Ringer's pen-strep solution using a P20 pipettor or a sterile glass Pasteur pipette rinsed with yolky Ringer's pen-strep solution. Store collected folds on ice while dissecting additional folds.After removing the culture dish from the incubator, use a pipettor or Pasteur pipette to remove the fibronectin solution from cover slips, dish, or well. After rinsing the fibronectin-coated substrate with Ringer's pen-strep solution, add an appropriate volume of complete culture media to the dish or well. To block the plastic and prevent the tissue from sticking, use a P20 or P200 pipettor, and rinse the pipette tip with yolky Ringer's pen-strep solution.Then, transfer the isolated neural folds towards the center of the fibronectin-coated cover slip, taking care to transfer as little Ringer's pen-strep solution as possible. After allowing the explants to settle for 10 to 15 minutes, place them into a humidified chamber at 38 degrees Celsius by slowly and carefully carrying the culture dish with plated neural folds. Remove the culture media with a Pasteur pipette, and rinse the wells with filter-sterilized PBS.Add 4%paraformaldehyde, and keep it on a platform shaker for 15 minutes at room temperature. At the end of the incubation, remove paraformaldehyde, rinse the wells three times with PBS, and add PBST containing 5%serum. Now, pipette 30 microliters of 200-nanomolar Oregon Green conjugated phalloidin onto a smooth surface for each cover slip.Using a pair of forceps, remove the cover slip from the PBST containing 5%serum, ensuring the orientation of the cells facing upward. Wick off excess liquid by briefly touching the cover slip's edge to a delicate task wiper. Gently place each cover slip cell side down onto the drop of diluted phalloidin, and incubate for 30 minutes at room temperature.After the incubation period, lift the cover slip from the staining solution and place it back into the culture dish, flipping it over so the cell side is up. Ensure to cover the cover slip with PBST, and place it on a platform shaker for 10 minutes, keeping the cover slips covered and in the dark. Remove PBST, and repeat washing the cover slips twice for a total of three 10-minute washes.Place one drop of mounting media onto a microscope slide. Mount the cover slip cell side down by slowly lowering the cover slip at an angle onto the mounting media to avoid creating bubbles, and allow the media to set before imaging. Finally, image the stained cells, and export them as TIFF files.The neural crest cells began to emerge from adherent neural fold explants within three to four hours of incubation, and migration was completed after approximately 20 hours. The migratory neural crest cells were labeled using HNK-1, and most cells appeared to be HNK-1 positive. The migrated neural crest cells were visualized by staining filamentous actin with phalloidin.Images of the phalloidin-stained cells were processed using ImageJ to produce a threshold image in which cells appeared black with a white background. The threshold image was analyzed and various measurements displayed in contrasting bar graphs or violin plots. For example, the average area of the 69 cells analyzed was approximately 802.11 square micrometers, ranging from 60.27 to 2, 664.53 square micrometers.Circularity reflects the cell's protrusiveness and ranges from 0.101 to 0.875. The lower values indicate an elongated shape, while a value of one indicates a perfect circle. Most cells exhibited an elongated shape, which is most easily seen in the violin plot.Migratory neural crest cells in this field had an average aspect ratio of approximately 2.13, and an aspect ratio of one indicates a symmetrical shape. Careful excision and explant plating are crucial for successful cultures. Allow neural folds to settle cut side down for 10 to 15 minutes, and then slowly transfer to the incubator to encourage adhesion.Variations on these techniques include transient genetic manipulation prior to culturing and time-lapse imaging during incubation. Additionally, this protocol allows for collection of premigratory and migratory neural crest cell populations for omics-level analysis.

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Developmental Biology

2.3K vistas.  Hamline University. Las células de la cresta neural emigran de los pliegues neurales craneales en embriones o cuando se colocan en cultivo. Esto proporciona un método conveniente para aislar las células primarias de la cresta neural migratoria sin disociación celular. Mientras que la migración de la cresta neural ocurre típicamente dentro del entorno embrionario tridimensional, la cultura bidimensional permite la visualización e investigación de los procesos relacionados con la migración.Aunque e...