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 KH2PO4 (see Table of Materials). Adjust pH to 7.4 and filter sterilize into 100 mL bottles. Store in a clean, dry location. Do not wash bottles with soap (treat as tissue culture glassware).</li>
	<li>Prepare 1 mg/mL stock solution fibronectin (FN) (see Table of Materials) by dissolving FN in sterile Ringer&#39;s solution and store in 100 &#181;L aliquots at -80 &#176;C (stable for at least 4 years).</li>
	<li>Prepare complete culture media by supplementing L15 media with 0.8% L-glutamine, 0.08% penicillin/streptomycin, 10% FBS, and 10% chick embryo extract12. Create 3 mL aliquots and store at -20 &#176;C or -80 &#176;C. 
	NOTE: If cultures are incubated in a CO2 incubator, DMEM/F12 needs to be substituted for L15, which is buffered for air.</li>
	<li>Prepare 4% paraformaldehyde solution in 1x PBS. Adjust pH to 7.4. Create 10 mL aliquots and store them at -20 &#176;C. 
	CAUTION: Paraformaldehyde must be handled in a fume hood.</li>
	<li>Prepare filter paper support frames (approximately 1 cm x 1.5 cm rectangles of paper with two or three punched holes overlapping on the long axis).
	<ol>
		<li>Punch holes along the edge of a large piece of filter paper using a standard three-hole punch, cut/trim the punched edge into a strip, and then cut the strip between the holes into rectangles ~ 1.5 cm in length. Autoclave filter papers before use (optional).</li>
	</ol>
	</li>
	<li>Gather dissection tools, including fine forceps (#5), blunt forceps, dissection scissors, spring scissors, and sharpened tungsten needles16.</li>
</ol>
2. Embryo incubation
<ol>
	<li>Obtain fertilized eggs from any of the sources mentioned above.</li>
	<li>Place the fertilized eggs in an upright position (long axis of the egg vertical with the pointed end down/blunt side up) inside a humidified incubator at 38 &#176;C (100 &#176;F).</li>
	<li>Incubate until four to seven somites have formed (stages 8+-9)17, which takes approximately 35 h. 
	NOTE: Incubation time can vary significantly depending on the strain, age of hens, season, etc., and must be experimentally determined. Programmable outlet timers are useful for initiating incubation during the night to obtain desired timing.</li>
	<li>After removing from the incubator, thoroughly spray the eggs with 70% ethanol and let them dry to disinfect the shells. 
	&#8203;NOTE: Embryos can be collected immediately, but letting eggs cool to room temperature decreases the fragility of the yolks and embryo loss during collection.</li>
</ol>
3. Preparation of culture dishes
<ol>
	<li>Select neural fold culture dishes (see Table of Materials) appropriate for the downstream application.
	<ol>
		<li>For cultures that will be fixed and stained, use glass coverslips placed into multi-well tissue culture dishes. 
		NOTE: Coverslips that match the well size will move less with fluid mixing, making explants less likely to shift and more likely to adhere to the coverslip rather than the dish bottom.</li>
		<li>Select glass-bottom single chamber or divided chamber dishes (see Table of Materials) for live imaging with an inverted microscope.</li>
		<li>For live imaging on an upright or stereo microscope, use 6-, 12-, or 24-well optically appropriate tissue culture plates.</li>
		<li>Select plastic tissue culture dishes for the mass collection of migratory neural crest cells for high-throughput analyses.</li>
	</ol>
	</li>
	<li>Add 10 U/mL of penicillin and 10 &#181;g/mL of streptomycin to a 100 mL bottle of sterile Ringer&#39;s solution (for example, 100 &#181;L of 10,000 U/mL penicillin and 10,000 &#181;g/mL streptomycin, to make Ringer&#39;s P/S). Use Ringer&#39;s P/S within 1 week.</li>
	<li>Thaw an aliquot of 1 mg/mL of FN on ice. Dilute with Ringer&#39;s P/S to a concentration of 10-100 &#181;g/mL of FN.</li>
	<li>Pipet enough FN solution to cover the bottom of the well or dish. For example, 100 &#181;L per well in a 24-well plate, 500 &#181;L for a 35 mm dish.</li>
	<li>Replace the lid and incubate dishes or plates with FN solution in a humidified incubator (or a covered tray with distilled water-soaked paper towels) at 38 &#176;C (100 &#176;F) for at least 1 h while dissecting neural folds.</li>
</ol>
4. Isolation of chick embryos
<ol>
	<li>Take care to maintain the orientation of the egg (the embryo floats to the top of the yolk during incubation). Use scissors or blunt forceps to poke a small hole in the shell, about 1/4-1/3 of the way down the length of the egg.
	<ol>
		<li>Carefully insert the tip of scissors or blunt forceps into the small hole, ensuring not to disrupt the yolk, and cut through the eggshell around the egg, removing the top of the eggshell (Figure 2A).</li>
	</ol>
	</li>
	<li>Position the embryo for isolation. 
	NOTE: If the embryo is not ideally located in the shell cup, use blunt forceps to carefully turn the yolk, so the embryo is on the top. Alternatively, pour the yolk into a gloved, cupped hand, being careful not to break the yolk and allowing the albumin to drain away (Figure 2B). Then, the embryo can be positioned by moving the yolk from hand to hand.</li>
	<li>Prepare the embryo by gently using the flat edge of closed blunt forceps to wipe away any excess albumin that remains on the yolk&#39;s surface covering the embryo (Figure 2C). Excess albumin can also be removed using gentle pressure with a delicate task wiper. 
	NOTE: Once albumin is cleared away, the yolk&#39;s surface should appear textured instead of smooth. Failure to wipe away sufficient albumin will inhibit the adhesion of the filter paper support in section 4, step 4.</li>
	<li>Use forceps to place a filter paper support frame over the embryo, with the embryo in the frame&#39;s window. Gently press down on the filter paper to adhere it onto the yolk.</li>
	<li>Cut around the outside of the filter paper frame with dissection scissors (Figure 2D). Use forceps or scissor tips to grasp the edge of the frame and gently lift the embryo away from the yolk (Figure 2E). Lifting away at an angle will help remove the embryo cleanly from the yolk.</li>
	<li>Place the embryo with the paper frame side down (embryo ventral side up) into a 60 mm or 100 mm Petri dish filled with Ringer&#39;s P/S (Figure 2F). Keep the dish of embryos on ice if collecting for an RNA- or protein-sensitive downstream application. 
	&#8203;NOTE: Failure to place embryos ventral side up risks detaching them from their paper frames. Multiple embryos can be collected and stored in Ringer&#39;s P/S for up to 1 h before moving to the neural fold dissection steps.</li>
</ol>
5. Dissecting neural folds
<ol>
	<li>Transfer an embryo to a clean dish containing Ringer&#39;s P/S solution and rinse the embryo by holding the filter paper frame with forceps and gently swishing back and forth to clear away any yolk that obscures the view of the embryo. Exchange the Ringer&#39;s P/S or transfer to a fresh dish if it becomes cloudy.</li>
	<li>Position the embryo dorsal/frame side up under a dissecting microscope. Leaving the embryo on the paper frame to keep it stretched taut and held in place, remove the vitelline membrane using forceps to expose the neural folds. 
	NOTE: If the embryo falls off the paper frame, it can be laid out flat in the dish or pinned to a sylgard coated dish18 for dissection.</li>
	<li>Using spring scissors or a sharpened tungsten needle, carefully excise the midbrain neural folds. Include tissue caudal to the expanding optic vesicles and rostral to the hindbrain, where rhombomere constrictions are just beginning to appear (the cardiac crescent is also a useful indicator, Figure 3B,C). Take care to excise the dorsal-most aspect of the neural fold with minimal contaminating neural tube and non-neural ectoderm (Figure 3C).</li>
	<li>Transfer the neural folds to a clean dish containing Ringer&#39;s P/S using a P20 pipettor or a sterile, glass Pasteur pipette rinsed with yolky Ringer&#39;s P/S (this blocks the plastic or glass to prevent the tissue from sticking). Store collected folds on ice while dissecting additional folds.</li>
</ol>
6. Plating neural folds
<ol>
	<li>Thaw an aliquot of complete culture media (section 1, step 3). Add 100 U/mL of penicillin and 100 &#181;g/mL of streptomycin and filter sterilize. Keep prepared media at 37-38 &#176;C while performing other steps.</li>
	<li>Remove culture dishes from the incubator (section 3, step 5). Using a pipettor or Pasteur pipette, remove the FN solution from coverslips, dishes, or wells. After rinsing the FN-coated substrate with Ringer&#39;s P/S, add an appropriate volume of complete culture media to the dishes or wells (500 &#181;L for wells in a 24-well plate, 2 mL for a 35 mm dish or a six-well plate). 
	NOTE: Smaller volumes can preserve expensive reagents (e.g., as low as 200 &#181;L for a 24-well plate) but ensure that the dish is sufficiently humidified to avoid evaporation.</li>
	<li>Using a p20 or p200 pipettor, first, rinse the pipette tip with yolky Ringer&#39;s P/S to block the plastic and prevent the tissue from sticking. Then, transfer the isolated neural folds onto the FN coated coverslips, taking care to transfer as little Ringer&#39;s P/S as possible. Place the fold(s) toward the center of the FN-coated coverslip. 
	NOTE: One or a few neural folds can be plated on a 12 mm coverslip in a 19 mm well, up to 50 on a 35 mm plate.</li>
	<li>After allowing the explants to settle for 10-15 min, place them into a humidified chamber at 38 &#176;C (100 &#176;F) by slowly and carefully carrying the culture dishes with plated neural folds. This will minimize the shifting of neural folds in the wells, ensuring they remain dispersed and adhere to any coverslips. 
	NOTE: When using L15 (not DMEM), explant culture dishes can also be incubated in an egg incubator using a covered tray with moistened paper towels.</li>
	<li>Incubate neural fold cultures in the humidified incubator for the duration of active migration (about 16-20 h total, Figure 4).</li>
</ol>
7. Fixing and staining of cultured migratory NCCs for morphological analysis
<ol>
	<li>Remove the culture media with a Pasteur pipette and rinse the wells with filter-sterilized 1x PBS.</li>
	<li>Add 4% paraformaldehyde for 15 min at room temperature. 
	NOTE: Fixation time may need to be experimentally determined. It is also possible to add paraformaldehyde directly to media for 10 min (50:50), remove, and replace with undiluted 4% paraformaldehyde for 10 min to retain more delicate cellular structures.</li>
	<li>Remove paraformaldehyde and rinse three times with 1x PBS.</li>
	<li>Stain fixed NCCs with an appropriate dye for morphological assessment. As an example, staining with Oregon Green conjugated phalloidin is detailed here. 
	NOTE: Visualizing the actin cytoskeleton with phalloidin19 reveals the structural complexity of migratory NCC with relatively uniform staining intensity; however, phalloidin may not highlight all cell areas. Dyes that label the plasma membrane (e.g., wheat germ agglutinin or DiI) or cytoplasm can also be used, but complicate the imaging of fine protrusions relative to the brightly staining cell body. In addition, immunofluorescence can be performed concurrently to label neural crest cells with HNK-1 or to determine the subcellular localization of a protein of interest7,20.
	<ol>
		<li>Remove the final PBS rinse and add PBS + 0.5% Triton X-100 (PBST) + 5% serum (FBS or from another animal appropriate for costaining with an antibody) to cover the coverslips and incubate for 10 min on a platform shaker at room temperature.</li>
		<li>Pipet 200 nM of Oregon Green conjugated phalloidin (diluted in PBST + 5% serum) onto a smooth surface (such as flexible paraffin sealing film) for each coverslip. For a 12 mm coverslip, pipette a 30 &#181;L volume.</li>
		<li>Using a pair of forceps, remove the coverslip from the PBST + 5% serum, ensuring to maintain the orientation of the cells facing upward. Briefly touch the edge of the coverslip to a delicate task wiper to wick off excess liquid.</li>
		<li>Gently place each coverslip cell side down onto the drop of diluted phalloidin. Incubate for 30 min at room temperature. Keep the coverslips in the dark during staining (cover with an aluminum foil-covered dish or place in a drawer).</li>
		<li>During the incubation, add PBST to the wells of the culture dish (750 &#181;L of PBST for each well of a 24-well plate).</li>
		<li>After the incubation period, lift coverslips off of the staining solution and place them back into the culture dish, flipping the coverslip over so the cell side is up. Ensure the coverslip is covered with PBST and place on a platform shaker for 10 min, keeping the coverslips covered and in the dark. Remove PBST and repeat this step two times, for a total of three 10 min washes.</li>
		<li>Place one drop (per coverslip) of mounting media (see Table of Materials) onto a microscope slide. 25 &#181;L volume works well for a 12 mm coverslip.</li>
		<li>After the final wash with PBST, touch the edge of the coverslip to a delicate task wiper to remove excess liquid, keeping track of the cell side of the coverslip.</li>
		<li>Mount the coverslip cell side down by slowly lowering the coverslip at an angle onto the mounting media to avoid creating bubbles. Allow the media to set before imaging.</li>
	</ol>
	</li>
</ol>
8. Morphological assessment of cultured migratory NCCs
<ol>
	<li>Image stained cells and export as .tiff files. 
	NOTE: A 40x objective works well for imaging cultures, but objectives ranging from 10x (to capture a whole field of migratory NCCs) to 100x (single NCCs) can be used to collect images for morphological assessment. Images in the present study were captured using an inverted multimodal imaging platform (see Table of Materials).</li>
	<li>Upload the images into an image analysis software (ImageJ21, see Table of Materials). Click on Image &#62; Duplicate to create a second copy of each image to use for analysis, thus leaving the original unedited, as most image processing cannot be reversed.</li>
	<li>Click on Image &#62; Adjust &#62; Brightness/Contrast and use sliding bars to adjust the brightness or contrast of the images.</li>
	<li>Convert the images to grayscale following the steps below.
	<ol>
		<li>If images are exported in RGB, they will upload in RGB as a merged channel. To separate the images, click on Image &#62; Color &#62; Split Channels to acquire single-channel grayscale images.</li>
		<li>If images are already separated, go to Image &#62; Type &#62; 8-bit to convert file to grayscale.</li>
	</ol>
	</li>
	<li>Click on Image &#62; Adjust &#62; Threshold to convert to a binary image where pixels are identified as cells (foreground; black) or background (white). Use the sliding bar and/or click on Auto to select the Threshold setting that clearly defines cells from the background.
	<ol>
		<li>If cells are overlapping or staining is not continuous, it may be necessary to process further using the Watershed or Fill Holes functions22. This will separate cells from one another or fill in gaps within cells to be analyzed. Click on Process &#62; Binary &#62; Make Binary to first convert the image into an 8-bit binary format. Then click on Process &#62; Binary &#62; Watershed or Fill Holes. 
		NOTE: The software will make its best fit for where signals need to be separated or filled in, which can be adjusted further using plug-ins if necessary.</li>
	</ol>
	</li>
	<li>Select the measurements to be captured. Click on Analyze &#62; Set Measurements and check the Shape Descriptors boxes for analysis of Circularity (Circ.), Aspect Ratio (AR), Roundness (Round), and Solidity. 
	NOTE: Other measurements may also be included, like Area and Perimeter, depending on the type of analysis needed.</li>
	<li>Click on Analyze &#62; Analyze Particles, and set the parameters from the &#34;Analyze Particles&#34; menu.
	<ol>
		<li>Under &#34;Size&#34;, roughly estimate how large the cells or particles of interest are in pixels, as background signal or smaller cell debris will also be included if the Size parameter remains set at 0-Infinity (for example, set as 500-Infinity in Figure 5). 
		NOTE: Adjusting the Size to a tighter range can exclude smaller or larger particles than the ones needed for analysis.</li>
		<li>Under &#34;Circularity&#34;, leave at 0-1.0 to measure all cell shapes in the image.</li>
		<li>Under &#34;Show&#34;, select Bare Outlines from the drop-down menu to inspect the cell outlines selected with the &#34;Analyze Particles&#34; function. Scan the outlines to ensure that overlapping cells are distinguished, but cells are not divided unnecessarily. Also, check the menu boxes for &#34;Display Results&#34;, &#34;Summarize&#34;, and &#34;Add to Manager&#34;.</li>
	</ol>
	</li>
	<li>Once all the parameters have been set, click on Ok. 
	NOTE: Four separate windows will pop up, showing (1) the bare outlines of the cells identified in the image, (2) the cells counted in the image, (3) the results of each cell&#39;s measurements, and (4) a summary of the total number of cells counted and their averaged measurements.
	<ol>
		<li>If debris was counted, overlapping cells were counted as one, single cells were counted as multiples, or many cells were omitted from the count, go back to the original, unedited image, make another duplicate, and adjust &#34;Threshold&#34; or other parameters.</li>
	</ol>
	</li>
</ol>

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(1360), 240-250 (1990).</li><li>Haupt, A., Minc, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+cells+sense+their+own+shape+-+mechanisms+to+probe+cell+geometry+and+their+implications+in+cellular+organization+and+function.">How cells sense their own shape - mechanisms to probe cell geometry and their implications in cellular organization and function.</a> Journal of Cell Science. 131 (6), (2018).</li><li>Ezin, M., Fraser, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+11+time-lapse+imaging+of+the+early+avian+embryo.">Chapter 11 time-lapse imaging of the early avian embryo.</a> Methods in Cell Biology. 87, 211-236 (2008).</li><li>Kulesa, P. M., Bailey, C. M., Cooper, C., Fraser, S. 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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+cranial+neural+crest+cells+release+extracellular+vesicles+that+are+critical+for+their+migration.">Chick cranial neural crest cells release extracellular vesicles that are critical for their migration.</a> Journal of Cell Science. , (2022).</li><li>Williams, R., Sauka-Spengler, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+ovo+electroporation+of+early+chicken+embryos.">Ex ovo electroporation of early chicken embryos.</a> STAR Protocols. 2 (2), 100424 (2021).</li><li>Moulton, J. 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. M., 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=Reconstruction+of+the+Global+Neural+Crest+Gene+Regulatory+Network+In+Vivo.">Reconstruction of the Global Neural Crest Gene Regulatory Network In Vivo.</a> Developmental Cell. 51 (2), 255-267 (2019).</li></ol>

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 ...

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Developmental Biology

2.3K Views.  Hamline University. 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. 

Video: Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3&#8217;UTRs by dual reporter plasmid transfection and dual luciferase assay.

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, fluid and nutrient supply). Consequently both the nervous and vascular systems develop alongside each other and share striking similarities in their branching architecture. Here we report embryonic manipulations that allow us to study the simultaneous development of neural crest-derived nervous tissue (in this case the enteric nervous system), and the vascular system. This is achieved by generating chicken chimeras via transplantation of discrete segments of the neural tube, and associated neural crest, combined with vascular DiI injection in the same embryo. Our method uses transgenic chickGFP embryos for intraspecies grafting, making the transplant technique more powerful than the classical quail-chick interspecies grafting protocol used with great effect since the 1970s. ChickGFP-chick intraspecies grafting facilitates imaging of transplanted cells and their projections in intact tissues, and eliminates any potential bias in cell development linked to species differences. This method takes full advantage of the ease of access of the avian embryo (compared with other vertebrate embryos) to study the co-development of the enteric nervous system and the vascular system.

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

Here we report dual labeling of neural crest cells and blood vessels using chickGFP neural tube intraspecies grafting combined with intra-vascular DiI injection. This experimental technique allows us to simultaneously visualize and study development of the NCC-derived (enteric) nervous system and the vascular system, during organogenesis.

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, ...

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to adequate numbers prior to transplantation. Identifying approaches to refine this process, to augment the production of all CNS cell types (i.e., neurons), and to possibly contribute to therapeutic cell therapy protocols is a high research priority. This report describes an easily applied in vivo &#8220;pre-conditioning&#8221; stimulus which can be delivered to awake, non-anesthetized animals. Thus, it is a non-invasive and non-stressful procedure. Specifically described are the procedures for exposing mouse or rat pups (aged postnatal day 1-8) to a brief (40-80 min) period of intermittent hypoxia (AIH). The procedures included in this video protocol include calibration of the whole-body plethysmography chamber in which pups are placed during AIH and the technical details of AIH exposure. The efficacy of this approach to elicit tissue-level changes in the awake animal is demonstrated through the enhancement of subsequent in vitro expansion and neuronal differentiation in cells harvested from the subventricular zone (SVZ). These results support the notion that tissue level changes across multiple systems could be observed following AIH, and support the continued optimization and establishment of AIH as a priming or conditioning modality for therapeutic cell populations.

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

This article describes the methodology for administering short periods of intermittent hypoxia to postnatal day 1-8 mouse or rat pups. This approach effectively elicits a robust tissue level &#8220;priming effect&#8221; on cultured neural progenitor cells that are harvested within 30 min of hypoxia exposure.

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to ...

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &amp;#8212; A High-throughput Screen in ES Cells

Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.

Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental neurobiology. Rather than use an animal model or genetically modified induced pluripotent cells, human neural stem cells provide an effective in vitro system to examine the effects of treatments, screen drugs, or examine individual differences. Here, we provide the detailed protocols for methods used to expand human fetal brain neural stem cells in culture with serum-free media, to differentiate them into various neuronal subtypes and astrocytes via different priming procedures, and to freeze and recover these cells. Furthermore, we describe a procedure of using human fetal brain neural stem cells to study Zika virus infection.

This article details the methods that are used to expand human fetal brain neural stem cells in culture, as well as how to differentiate them into various neuronal subtypes and astrocytes, with an emphasis on the use of neural stem cells to study Zika virus infection.

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...