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. Day 1: Isolation of trunk neural tubes for overnight culture on coverslips
<ol>
	<li>Incubate chick eggs for 56 h at 38&deg; C. Remove the eggs from incubation, mildly spray them with 70% Ethanol, and then allow them to dry. Break the eggs open into a UV-sterilized glass tray.</li>
	<li>Extract each embryo from its yolk and place it in chick Ringer's. Do this by first cutting around its blood islands with curved scissors; then, with blunt forceps, pick the embryo up by its extraembryonic membrane and place it in a sterile plastic Petri dish containing chick Ringer&prime;s solution.</li>
	<li>Isolate the trunk of each embryo by trimming off excess extraembryonic membranes as well as cranial, vagal, and sacral axial levels using a tungsten needle (Fig. 1). First, select about 9 embryos that are between stages HH15-17 11. For stages HH15 and up, the forebrain and hindbrain axes form an acute angle and therefore the head appears to tilt caudally. At stage HH17, the tail bud is present and tilts ventrally but does not yet contain somites. With a tungsten needle, trim off extraembryonic membranes to about 2 mm from the embryo and cut off any embryonic tissues anterior to somite 10. Also remove all caudal embryonic tissues starting from around the fifth most newly formed somite.</li>
	<li>Place the isolated embryonic trunks in Dispase (0.24 U/ml DMEM) and incubate them for 1 h 15 min at 37&deg; C and 5% CO2. Once the trunks begin incubation, start preparing 6 coverslips (CS) for culturing NT explants (steps 1.5-1.8).</li>
	<li>Rinse 6 CS in 70% ethanol (diluted in sterile, ultrapure water), and then allow them to dry. Using a lab marker, draw a circle in the center of each CS that is about 1 cm in diameter (this circle will later help you identify where a fibronectin coat has been applied). On the same face of each CS, write the word "be" (or some other asymmetrical word or shape) outside the drawn circle (this will help you identify whether the marked side of the CS is facing up or down).</li>
	<li>Place each CS in a separate 40 x 10 mm sterile dish with the marked surface facing down and allow the dish to sit open underneath a germicidal UV lamp for 10 min.</li>
	<li>Apply 60 &mu;l fibronectin (FN; 10 &mu;g/ml DMEM) to the unmarked surface of the CS while making sure the entire area within the 1 cm circle is coated. Place the dishes to incubate at 37&deg; C for 30 min and then carefully aspirate the fibronectin from each CS.</li>
	<li>Add 250 &mu;l "culture" medium [DMEM with L-Glutamine (2 mM), Penicillin (100U/ml), Streptomycin (100 mcg/ml), and 8% fetal bovine serum (FBS)] to the FN-coated area of the CS. Place the dishes containing each CS at 37&deg; C and 5% CO2 again until the NTs have been isolated.</li>
	<li>Transfer all the incubated embryo trunks to one 5 cm glass Petri dish containing L15 medium and start dissecting out each NT using fine forceps and a tungsten needle (Fig. 1). Carefully slice along the border of the NT and somites with a sharp tungsten needle while being cautious not to damage the NT. It is often easier to begin isolating each NT from the caudal-most end of the trunk.</li>
	<li>Select 6 of the straightest and longest NTs to culture overnight (NTs between roughly 8 and 15 somites long are recommended). Using a micropipette tip primed with culture medium, transfer each of the 6 NTs to its own previously prepared CS (from steps 1.5 through 1.8). Be sure that the NT does not remain floating at the surface. If the NT is floating, drip medium onto it until it sinks using a micropipette.</li>
	<li>Place each dish at 37&deg; C and 5% CO2 overnight. Be careful to ensure each NT is within the FN-coated area of its respective CS immediately prior to placing the dish in the incubator (by using the circle drawn in step 1.5 as a reference). A micropipette can be used to better adjust the position of each NT if needed.</li>
	<li>Place at least 2 ml of culture medium (without serum) into a sterile 15 ml centrifuge tube and incubate overnight at 37&deg; C and 5% CO2. Leave the cap slightly unscrewed to allow the pH of the medium to adjust overnight. Pre-incubating the medium is important to help prevent bubble formation in your chamber, which may disrupt the establishment of a molecular gradient. Such "preincubated " medium should be used in all future steps. When not being used, this medium should be incubating at 37&deg; C.</li>
</ol>
2. Day 2: Loading the modified Zigmond chamber and time-lapse analysis of cell migration
<ol>
	<li>Out of the 6 NTs cultured, select the 3 cultures best suited for analysis. Generally, NCC cultures that have at least one long, straight edge should be selected (Fig. 2A). The 3 best cultures will be used to load and film 3 modified Zigmond chambers throughout the day, each with a different treatment. Out of the 3 cultures, choose one for loading the first chamber and return the others to the incubator for later use.</li>
	<li>Using a cotton swab, apply a thin, even layer of petroleum jelly surrounding the reservoirs and bridge of one modified Zigmond chamber.</li>
	<li>With a tungsten needle, gently remove the NT from the CS, while leaving the surrounding NCCs attached to the surface of the CS. Mark the dish with a pen to remember the orientation of the straightest edge of the NCC culture.</li>
	<li>Place a few drops of preincubated medium onto the bridge. Pick up the CS with fine forceps, dab the edge of the CS against a Kimwipe to remove most of the old culture medium, then immediately place the CS on the modified Zigmond chamber so that the straight edge of the culture to be filmed is centered over the length of the bridge and roughly perpendicular to the bridge-reservoir border (Fig. 2A,B).</li>
	<li>Using an inverted microscope, move the straight NCC border to be on the side of the bridge closest to the reservoir that will contain the suspected chemotactant (Fig. 2B; for controls this will correspond to whichever reservoir is loaded second). Also, more finely align the straight edge of the culture to be perpendicular to the bridge-reservoir border.</li>
	<li>Carefully, but securely press the CS into the petroleum jelly present on the Zigmond chamber, making sure it is completely sealed on to the chamber, then place additional petroleum jelly along the edge of the CS to further ensure it will be airtight. Fine-adjust the angle of the NCC border again to correct for any movement during the sealing process.</li>
	<li>Load the reservoir that will not contain the suspected chemotactant first (Fig. 2B). Do so by loading a 1 ml syringe (25 G x 1.5 in. attached needle) with roughly 300 &mu;l preincubated medium and inject the medium into the reservoir until full (while being careful not to generate any bubbles in the reservoir). Plug the reservoir on both sides with a sufficient amount of petroleum jelly prior to loading the next reservoir.</li>
	<li>Repeat step 2.7, except this time using preincubated medium containing the candidate chemotactant. It is critical when generating a molecular gradient across the culture to always load the reservoir containing the molecule to be tested after loading the reservoir lacking the tested molecule.</li>
	<li>Place the loaded Zigmond chamber at 37&deg; C to incubate for 1 h prior to filming. Image the straightest border of the NCC culture for 3 h at 90 s intervals while incubating at roughly 37&deg; C (Fig. 2A,B). Prior to creating any movie, be sure to align the camera so that the edge of the images to be obtained are aligned with and touching the edge of the bridge that borders the last reservoir loaded (Fig. 2B, upper panel; dashed box represents ideal position for imaging). This will facilitate later software analysis by standardizing the directionality of the molecular gradient applied and the distance from the reservoir filmed in each movie produced.</li>
	<li>For controls, repeat steps 2.2-2.9 for each of the two other NCC cultures selected (in step 2.1), but fill each reservoir with an appropriate medium. For one type of control fill both reservoirs with preincubated medium not containing the molecule to be tested. For a second control treatment, prime the bridge with a few drops of preincubated medium containing the suspected chemotactant prior to mounting the CS. Then, load both reservoirs with the same medium containing the suspected chemotactant.</li>
	<li>Use ImageJ (NIH) Manual Tracking <a href="http://rsb.info.nih.gov/ij/plugins/track/track.html">(rsb.info.nih.gov/ij/plugins/track/track.html)</a> and Chemotaxis and Migration Tool v1.01 <a href="http://www.ibidi.de/applications/ap_chemo.html">(www.ibidi.de/applications/ap_chemo.html)</a> plugins to track the migration of peripheral NCCs along the straight border of the culture just imaged and to analyze various parameters of the migratory trajectories obtained (Fig. 2B-C).</li>
</ol>
3. Representative Results:
A sample of cell trajectories from a movie where many trunk NCCs were responsive to a candidate chemoattractant using the above technique is shown (Fig. 2D). Most cells in this example of a positive response displayed a net movement up the chemoattractant gradient (as shown in red). Trajectory data can be used to analyze other properties of cell migration as well.
In order to visually evaluate an applied gradient in a modified Zigmond chamber, an Alexa Fluor 488 IgM conjugate (MW ~ 900 kDa) was loaded into the second reservoir of a modified Zigmond chamber (at roughly 40 &mu;g/ml H2O). A gradient was established by 1 h and still somewhat present after 26 h, but greatly diminished by 50 h (Fig. 3). If the molecule to be tested is smaller, then the applied gradient will degrade faster than what is shown.
<img alt="Figure 1" src="/files/ftp_upload/3330/3330fig1.jpg" /> 
Figure 1. Explantation of trunk-level NTs for overnight culturing on fibronectin-coated coverslips. Because trunk NCCs delaminate from the dorsal NT located adjacent to somites 8-28, this segment of the NT is isolated by microdissection and cultured overnight on a fibronectin-coated CS to allow for emigration of NCCs from the NT explant. Isolated NTs that are between 8-15 somites long and relatively straight are best suited for overnight culturing as they tend to yield NCC cultures with longer straight borders. Regions of the neural tube that give rise to other neural crest axial levels are shown in a smaller font. s, somite.
<img alt="Figure 2" src="/files/ftp_upload/3330/3330fig2.jpg" /> 
Figure 2. Method for evaluating the migration of explanted trunk NCCs using a modified Zigmond chamber. (A) Elongated trunk NCC cultures are prepared by overnight culturing of NTs and resultant NCC cultures with at least one long, straight border are selected for experimentation. The longest straight border of a selected culture is then positioned perpendicular to the bridge-reservoir border, and therefore parallel to the vector of the future applied gradient. (B) After fine-adjusting the position of the NCC culture on the Zigmond chamber and sealing the coverslip to the chamber, the chamber is loaded. When testing chemotaxis, the reservoir that will not contain the suspected chemotactant (-) is loaded first and sealed. Then, the other reservoir is loaded with the suspected chemotactant (+) and sealed. Peripheral NCCs along the previously selected border can then be imaged and tracked using the Manual Tracking plugin for ImageJ (bottom panel). (C) Numerous migratory characteristics in response to the applied gradient can be assessed based on tracking data. For instance, a chemotaxis index can be derived by dividing a cell's displacement along the x-axis by the total distance it has migrated. (D) An example of an attractive response is shown by a cell trajectory plot initially generated by the Chemotaxis and Migration Tool plugin for ImageJ. The starting point of each trajectory is set to the origin (0,0). Note how many more cells migrate toward the chemoattractant source.The center of mass of all cells at their final position (blue cross; all cells equally weighted) is also closer to the chemoattractant source. NCCs, neural crest cells; Red tracks, cells that migrated toward the reservoir loaded with a suspected chemoattractant; Black tracks, cells that migrated away; (+), higher chemotactant concentration; (-), lower chemotactant concentration.
<img alt="Figure 3" src="/files/ftp_upload/3330/3330fig3.jpg" /> 
Figure 3. Intensity profiles across the bridge of a modified Zigmond chamber at different times following the addition of an Alexa Fluor 488 IgM conjugate. The chamber was loaded in a similar manner to that described in the protocol with the main exceptions that preincubated water (instead of preincubated medium) was used to dilute the antibody to 40 &mu;g/ml, and small air pockets were present at the ends of the bridge (away from the slice where the above intensity profiles where taken). Initially, no gradient was present across most of the bridge. By 1 h a gradient was established and remained present through to 26 h. By 50 h the presence of the gradient was inconsistent at different areas of the bridge, and when present, the steepness of the gradient was greatly diminished. All profiles were generated from an identical slice across the bridge (from one bridge-reservoir border to the other) using AxioVision 4.6 software. Note that even while air pockets were present, the gradient was not disrupted. High, high intensity; Low, low intensity; x-axis, distance across entire width of bridge (2 mm); (+), reservoir loaded with the Alexa Fluor 488 IgM conjugate; (-), reservoir not loaded with the conjugate.
<img alt="Figure 4" src="/files/ftp_upload/3330/3330fig4.jpg" /> 
Figure 4. Modified Zigmond chamber specifications. Shown is a diagram of the modified Zigmond chamber used here along with its dimensional specifications (&plusmn;0.2 mm). Measurements can be moderately adjusted in order to match individual preferences.
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&Prime; 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&Prime;) 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&Prime;) 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&Prime;) and then traverse to 7.62 mm (0.300&Prime;) in the opposite (negative) direction to a complete reservoir length of 15.24 mm (0.600&Prime;). Offset the bit to 3.03 mm (0.119&Prime;) 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>

<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. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+direct-viewing+chemotaxis+chamber.">A new direct-viewing chemotaxis chamber.</a> Journal of Cell Science. 99, 769-775 (1991).</li></ol>

No conflicts of interest declared.

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><tbody><tr><td>DMEM</td><td>Omega Scientific</td><td>DM-22</td><td></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 &amp;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 H&lt;sub&gt;2&lt;/sub&gt;O 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></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&#039;s from egg yolk</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;Supplemental Protocol: Fabrication of a Modified Zigmond Chamber&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;Please refer to Figure 4 as a reference for the protocol below:&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;ol&gt; &lt;li&gt;Purchase a sheet of 3/16&quot; thick polished acrylic (4.45 mm actual thickness).&lt;/li&gt; &lt;li&gt;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.&lt;/li&gt; &lt;li&gt;Set the chamber blank on a vise. With a milling machine and a 6.35 mm (1/4&quot;) end mill bit, finish machining the sides of the chamber to their exact dimensions: 30.07 mm x 61.39 mm.&lt;/li&gt; &lt;li&gt;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.&lt;/li&gt; &lt;li&gt;Acquire the chamber height (z-axis) by touching the end mill bit to the top surface and zero the height.&lt;/li&gt; &lt;li&gt;Using a 3.91 mm (0.154&quot;) 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&quot;) and then traverse to 7.62 mm (0.300&quot;) in the opposite (negative) direction to a complete reservoir length of 15.24 mm (0.600&quot;). Offset the bit to 3.03 mm (0.119&quot;) along the x-axis (negative direction) and repeat the same process for the second reservoir.&lt;/li&gt; &lt;li&gt;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.&lt;/li&gt; &lt;li&gt;Soak the chamber well in warm soapy water to help remove any chemical contaminants.&lt;/li&gt; &lt;li&gt;Soak and rinse the chamber well in double-distilled water to remove any soap. The chambers are now ready to use as described above.&lt;/li&gt; &lt;/ol&gt;</td></tr></tbody></table>

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

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

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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.

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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Neuroscience

12.4K Views.  California State University, Northridge. 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.

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

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Developmental Biology

Migratory Behavior of Cells Generated in Ganglionic Eminence Cultures

Migration of cells is a common process that leads to the development and maturation of the vertebrate central nervous system (Hatten, '99). The cerebral cortex consists of two basic neuronal types: excitatory and inhibitory. These cells arise in distinct areas and migrate into the cortex along different routes (Pearlman et al., '98). Inhibitory interneurons migrate tangentially from subcortical sources, mostly from different regions of the ganglionic eminences (Gelman et al., '09; Xu et al., '04). Their movement requires precise spatiotemporal control imposed by environmental cues, to allow for the establishment of proper cytoarchitecture and connectivity in the cerebral cortex (Caviness &amp; Rakic, '78; Hatten, '90; Rakic, '90). To study the migratory behavior of cells generated in proliferative zones of the ganglionic eminences (GE) in newborn ferrets in vitro we used a 3 dimensional culture arrangement in a BD Matrigel Matrix. The culture setup consisted of two GE explants and a source of tested proteins extracted from the cerebral cortex and adsorbed on fluorescent latex Retrobeads IX positioned between the explants (Hasling et al., '03; Riddle et al., '97). After 2-3 days of culture, the cells start to appear at the edge of the explant showing a propensity to leave the tissue in a radial direction. Live imaging allowed observation of migratory patterns without the necessity of labeling or marking the cells. When exposed to fractions of the protein extract obtained from isochronic ferret cortex, the GE cells displayed different behaviors as judged by quantitative kinetic analysis of individual moving cells.

Time lapse imaging of 3D tissue culture allows studying migratory behavior of individual cells originating from ganglionic eminence in reaction to fractionated protein extract from cerebral cortex.

Migration of cells is a common process that leads to the development and maturation of the vertebrate central nervous system (Hatten, '99). The cerebral ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

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 ...

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

This protocol describes the dissection and culture of cranial neural crest cells from mouse models, primarily for the study of cell migration. We describe the live imaging techniques used and the analysis of speed and cell shape changes.

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Biology

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.

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 ...

In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration

Neural crest cells (NCCs) are highly migratory cells that originate from the dorsal region of the neural tube. The emigration of NCCs from the neural tube is an essential process for NCC production and their subsequent migration toward target sites. The migratory route of NCCs, including the surrounding neural tube tissues, involves hyaluronan (HA)-rich extracellular matrix. To model NCC migration into these HA-rich surrounding tissues from the neural tube, a mixed substrate migration assay consisting of HA (average molecular weight: 1,200-1,400 kDa) and collagen type I (Col1) was established in this study. This migration assay demonstrates that NCC cell line, O9-1, cells are highly migratory on the mixed substrate and that the HA coating is degraded at the site of focal adhesions in the course of migration. This in vitro model can be useful for further exploration of the mechanistic basis involved in NCC migration. This protocol is also applicable for evaluating different substrates as scaffolds to study NCC migration.

In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration

This protocol outlines an in vitro migration experiment suitable for the functional analysis of the molecules involved in the in vivo migration of neural crest cells into the hyaluronan-rich extracellular matrix.

Neural crest cells (NCCs) are highly migratory cells that originate from the dorsal region of the neural tube. The emigration of NCCs from the neural tube ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

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

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Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Migratory Behavior of Cells Generated in Ganglionic Eminence Cultures

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

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

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme