Name: a galvanotaxis assay for analysis of neural precursor cell migration kinetics in an externally applied direct current electric field
Uploaded: 2012-10-13T14:00:00
Description: Watch this Scientific Journal Video about A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field at JoVE.com

This work is funded by the Natural Sciences and Engineering Research Council of Canada (grant #249669, and #482986) and Heart and Stroke Foundation of Canada (grant #485508). The authors thank Youssef El-Hayek and Dr. Qi Wan for their assistance in developing the experimental protocols.

All procedures involving animal handling were approved by the University of Toronto Animal Care Committee in accordance with institutional guidelines (protocol no. 20009387). The following methods should be performed using sterile tools and techniques, in a laminar flow hood where applicable.
In the protocol text below, the phrase "EFH-SFM" refers to serum free media supplemented with epidermal growth factor, basic fibroblast growth factor and heparin. EFH-SFM is used when investigating the ga...

<ol>
	<li>Borgens, R. B., Shi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncoupling+histogenesis+from+morphogenesis+in+the+vertebrate+embryo+by+collapse+of+the+transneural+tube+potential.">Uncoupling histogenesis from morphogenesis in the vertebrate embryo by collapse of the transneural tube potential.</a> Dev. Dyn. 203, 456-467 (1995).</li><li>Song, B., Zhao, M., Forrester, J. V., McCaig, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+cues+regulate+the+orientation+and+frequency+of+cell+division+and+the+rate+of+wound+healing+in+vivo.">Electrical cues regulate the orientation and frequency of cell division and the rate of wound healing in vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 13577-13582 (2002).</li><li>Song, B., Zhao, M., Forrester, J., McCaig, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+regeneration+and+wound+healing+are+stimulated+and+directed+by+an+endogenous+electrical+field+in+vivo.">Nerve regeneration and wound healing are stimulated and directed by an endogenous electrical field in vivo.</a> Journal of Cell Science. 117, 4681-4690 (2004).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+and+population+analyses+demonstrate+that+an+EGF-responsive+mammalian+embryonic+CNS+precursor+is+a+stem+cell.">Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell.</a> Dev. Biol. 175, 1-13 (1996).</li><li>Chiasson, B. J., Tropepe, V., Morshead, C. M., vander Kooy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+mammalian+forebrain+ependymal+and+subependymal+cells+demonstrate+proliferative+potential,+but+only+subependymal+cells+have+neural+stem+cell+characteristics.">Adult mammalian forebrain ependymal and subependymal cells demonstrate proliferative potential, but only subependymal cells have neural stem cell characteristics.</a> J. Neurosci. 19, 4462-4471 (1999).</li><li>Erickson, C. A., Nuccitelli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+fibroblast+motility+and+orientation+can+be+influenced+by+physiological+electric+fields.">Embryonic fibroblast motility and orientation can be influenced by physiological electric fields.</a> J. Cell Biol. 98, 296-307 (1984).</li><li>Zhao, M., Agius-Fernandez, A., Forrester, J. V., McCaig, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orientation+and+directed+migration+of+cultured+corneal+epithelial+cells+in+small+electric+fields+are+serum+dependent.">Orientation and directed migration of cultured corneal epithelial cells in small electric fields are serum dependent.</a> J. Cell Sci. 109, 1405-1414 (1996).</li><li>Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct-Current+Electrical+Field+Guides+Neuronal+Stem/Progenitor+Cell+Migration.">Direct-Current Electrical Field Guides Neuronal Stem/Progenitor Cell Migration.</a> Stem Cells. 26, 2193-2200 (2008).</li><li>Song, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+direct+current+electric+fields+to+cells+and+tissues+in+vitro+and+modulation+of+wound+electric+field+in+vivo.">Application of direct current electric fields to cells and tissues in vitro and modulation of wound electric field in vivo.</a> Nat. Protoc. 2, 1479-1489 (2007).</li><li>Huang, C. -. W., Cheng, J. -. Y., Yen, M. -. H., Young, T. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrotaxis+of+lung+cancer+cells+in+a+multiple-electric-field+chip.">Electrotaxis of lung cancer cells in a multiple-electric-field chip.</a> Biosen Bioelectron. 24, 3510-3516 (2009).</li><li>SATO, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Input-output+relationship+in+galvanotactic+response+of+Dictyostelium+cells.">Input-output relationship in galvanotactic response of Dictyostelium cells.</a> Biosystems. 88, 261-272 (2007).</li><li>Meng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+mediated+electrotaxis+of+embryonic+and+adult+neural+progenitor+cells+in+the+presence+of+growth+factors.">PI3K mediated electrotaxis of embryonic and adult neural progenitor cells in the presence of growth factors.</a> Exp. Neurol. 227, 210-217 (2011).</li><li>Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+lipids,+EGF+receptors,+and+intracellular+signals+colocalize+and+are+polarized+in+epithelial+cells+moving+directionally+in+a+physiological+electric+field.">Membrane lipids, EGF receptors, and intracellular signals colocalize and are polarized in epithelial cells moving directionally in a physiological electric field.</a> The FASEB Journal. , (2002).</li><li>Fang, K., Ionides, E., Oster, G., Nuccitelli, R., Isseroff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+receptor+relocalization+and+kinase+activity+are+necessary+for+directional+migration+of+keratinocytes+in+DC+electric+fields.">Epidermal growth factor receptor relocalization and kinase activity are necessary for directional migration of keratinocytes in DC electric fields.</a> J. Cell Sci. 112, 1967-1976 (1999).</li><li>McCaig, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+Cell+Behavior+Electrically:+Current+Views+and+Future+Potential.">Controlling Cell Behavior Electrically: Current Views and Future Potential.</a> Physiol. Rev. 85, 943-978 (2005).</li><li>Morshead, C. M., Benveniste, P., Iscove, N. N., vander Kooy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+competence+is+a+rare+property+of+neural+stem+cells+that+may+depend+on+genetic+and+epigenetic+alterations.">Hematopoietic competence is a rare property of neural stem cells that may depend on genetic and epigenetic alterations.</a> Nat. Med. 8, 268-273 (2002).</li><li>Babona-Pilipos, R., Droujinine, I. A., Popovic, M. R., Morshead, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+Subependymal+Neural+Precursors,+but+Not+Differentiated+Cells,+Undergo+Rapid+Cathodal+Migration+in+the+Presence+of+Direct+Current+Electric+Fields.">Adult Subependymal Neural Precursors, but Not Differentiated Cells, Undergo Rapid Cathodal Migration in the Presence of Direct Current Electric Fields.</a> PLoS ONE. 6, e23808 (2011).</li><li>Deuschl, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Randomized+Trial+of+Deep-Brain+Stimulation+for+Parkinson's+Disease.">A Randomized Trial of Deep-Brain Stimulation for Parkinson's Disease.</a> N. Engl. J. Med. 355, 896-908 (2006).</li><li>Stone, S. S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+Entorhinal+Cortex+Promotes+Adult+Neurogenesis+and+Facilitates+Spatial+Memory.">Stimulation of Entorhinal Cortex Promotes Adult Neurogenesis and Facilitates Spatial Memory.</a> J. Neurosci. 31, 13469-13484 (2011).</li></ol>

This protocol has been adapted from the well-established methods of previous studies7-9. Galvanotactic chambers can be constructed using a variety of different techniques, including the construction of a separate glass well for confinement of cell seeding, or using CO2 laser ablation for microfabrication of the central trough10,11. Some techniques may be more laborious or costly than others. We have described a simple and cost-effective assay for constructing a NPC galvanotaxis chamber us...

<table border="1">
 <tbody>
 <tr>
 <td height="44">Name of item</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Neural Precursor Cell Isolation</td>
 </tr>
 <tr>
 <td>2M NaCl</td>
 <td>Sigma</td>
 <td>S5886</td>
 <td>11.688 g dissolved in 100 ml dH2O</td>
 </tr>
 <tr>
 <td>1M KCl</td>
 <td>Sigma</td>
 <td>P5405</td>
 <td>7.456 g dissolved in 100 ml dH2O</td>
 </tr>
 <tr>
 <td>1M MgCl2</td>
 <td>Sigma</td>
 <td>M2393</td>
 <td>20.33 g dissolved in 100 ml dH2O</td>
 </tr>
 <tr>
 <td>155 mM NaHCO3</td>
 <td>Sigma</td>
 <td>S5761</td>
 <td>1.302 g dissolved in 100 ml dH2O</td>
 </tr>
 <tr>
 <td>0.5M Glucose</td>
 <td>Sigma</td>
 <td>G6152</td>
 <td>9.01 g dissolved in 100 ml dH2O</td>
 </tr>
 <tr>
 <td>108 mM CaCl2 </td>
 <td>Sigma</td>
 <td>C7902</td>
 <td>1.59 g dissolved in 100 ml dH2O</td>
 </tr>
 <tr>
 <td>Penicillin-streptomycin</td>
 <td>Gibco</td>
 <td>15070</td>
 <td></td>
 </tr>
 <tr>
 <td>Bovine pancreas trypsin</td>
 <td>Sigma</td>
 <td>T1005</td>
 <td></td>
 </tr>
 <tr>
 <td>Sheep testes hyaluronidase</td>
 <td>Sigma</td>
 <td>H6254</td>
 <td></td>
 </tr>
 <tr>
 <td>Kynurenic acid</td>
 <td>Sigma</td>
 <td>K3375</td>
 <td></td>
 </tr>
 <tr>
 <td>Ovomucoid trypsin inhibitor</td>
 <td>Worthington</td>
 <td>LS003086</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM </td>
 <td>Invitrogen</td>
 <td>12100046</td>
 <td></td>
 </tr>
 <tr>
 <td>F12</td>
 <td>Invitrogen</td>
 <td>21700075</td>
 <td></td>
 </tr>
 <tr>
 <td>30% Glucose</td>
 <td>Sigma</td>
 <td>G6152</td>
 <td></td>
 </tr>
 <tr>
 <td>7.5% NaHCO3</td>
 <td>Sigma</td>
 <td>S5761</td>
 <td></td>
 </tr>
 <tr>
 <td>1M HEPES</td>
 <td>Sigma</td>
 <td>H3375</td>
 <td>23.83 g dissolved in 100 ml dH2O</td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Gibco</td>
 <td>25030</td>
 <td></td>
 </tr>
 <tr>
 <td>EGF</td>
 <td>Invitrogen</td>
 <td>PMG8041</td>
 <td>Reconstitute in 1 ml of hormone mix and aliquot into 20 &mu;l units.</td>
 </tr>
 <tr>
 <td>FGF</td>
 <td>Invitrogen</td>
 <td>PHG0226</td>
 <td>Reconstitute in 0.5 ml of hormone mix and aliquot into 20 &mu;l units.</td>
 </tr>
 <tr>
 <td>Heparin</td>
 <td>Sigma</td>
 <td>H3149</td>
 <td></td>
 </tr>
 <tr>
 <td>Apo-transferrin</td>
 <td>R&amp;D Systems</td>
 <td>3188-AT</td>
 <td>0.1 g dissolved into 4 ml dH20</td>
 </tr>
 <tr>
 <td>Putrescine</td>
 <td>Sigma</td>
 <td>P7505</td>
 <td>Dissolve 9.61 mg into Apo-transferrin solution</td>
 </tr>
 <tr>
 <td>Insulin</td>
 <td>Sigma</td>
 <td>I5500</td>
 <td>Dissolve 25 mg into 0.5 ml of 0.1N HCl and add to 3.5 ml of dH20</td>
 </tr>
 <tr>
 <td>Selenium</td>
 <td>Sigma</td>
 <td>S9133</td>
 <td></td>
 </tr>
 <tr>
 <td>Progesterone</td>
 <td>Sigma</td>
 <td>P6149</td>
 <td></td>
 </tr>
 <tr>
 <td>Standard Dissection Tools</td>
 <td>Fine Science Tools</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Dissection microscope</td>
 <td>Zeiss</td>
 <td>Stemi 2000</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Galvanotaxis Chamber Preparation</td>
 </tr>
 <tr>
 <td>Square glass cover slides</td>
 <td>VWR</td>
 <td>16004</td>
 <td></td>
 </tr>
 <tr>
 <td>6N Hydrochloric Acid</td>
 <td>VWR</td>
 <td>BDH3204-1</td>
 <td></td>
 </tr>
 <tr>
 <td>High vacuum grease</td>
 <td>Dow Corning</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>60 mm Petri dishes</td>
 <td>Fisher Scientific</td>
 <td>0875713A</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-L-lysine</td>
 <td>Sigma</td>
 <td>P4707</td>
 <td></td>
 </tr>
 <tr>
 <td>Matrigel</td>
 <td>BD Biosciences</td>
 <td>354234</td>
 <td>Thaw and aliquot into 150 &mu;l units</td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Invitrogen</td>
 <td>10082139</td>
 <td>Only use if inducing NPC differentiation, otherwise use SFM + EFH culture media as indicated above</td>
 </tr>
 <tr>
 <td>Counting microscope</td>
 <td>Olympus</td>
 <td>CKX41</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Live Cell Time-Lapse Imaging</td>
 </tr>
 <tr>
 <td>Silver wire</td>
 <td>Alfa Aesar</td>
 <td>11434</td>
 <td></td>
 </tr>
 <tr>
 <td>UltraPure Agarose</td>
 <td>Invitrogen</td>
 <td>15510-027</td>
 <td></td>
 </tr>
 <tr>
 <td>Heat Inactivated FBS</td>
 <td>Sigma</td>
 <td>16140071</td>
 <td></td>
 </tr>
 <tr>
 <td>PVC tubing</td>
 <td>Fisher Scientific</td>
 <td>80000006</td>
 <td>3/32"ID x 5/32"OD</td>
 </tr>
 <tr>
 <td>Bleach</td>
 <td>Clorox</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>10 cc syringe</td>
 <td>BD</td>
 <td>309604</td>
 <td></td>
 </tr>
 <tr>
 <td>18 gauge needle</td>
 <td>BD</td>
 <td>305195</td>
 <td></td>
 </tr>
 <tr>
 <td>Dremel drill</td>
 <td>Dremel</td>
 <td>Model 750</td>
 <td></td>
 </tr>
 <tr>
 <td>Inverted microscope equipped with humidified, incubated chamber</td>
 <td>Zeiss</td>
 <td>Axiovert-200M</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>
Recipes
<table border="1">
 <tbody>
 <tr>
 <td>Item</td>
 <td>Volume</td>
 </tr>
 <tr>
 <td>2M NaCl</td>
 <td>6.2 ml</td>
 </tr>
 <tr>
 <td>1M KCl</td>
 <td>0.5 ml</td>
 </tr>
 <tr>
 <td>1M MgCl2</td>
 <td>0.32 ml</td>
 </tr>
 <tr>
 <td>155mM NaHCO3</td>
 <td>16.9 ml</td>
 </tr>
 <tr>
 <td>1M Glucose</td>
 <td>1 ml</td>
 </tr>
 <tr>
 <td>108 mM CaCl2</td>
 <td>0.09256 ml</td>
 </tr>
 <tr>
 <td>Penicillin-streptomycin</td>
 <td>1 ml</td>
 </tr>
 <tr>
 <td>Autoclaved water</td>
 <td>74 ml</td>
 </tr>
 </tbody>
</table>
Artificial cerebrospinal fluid
<table border="1">
 <tbody>
 <tr>
 <td>Item</td>
 <td>Volume or Mass</td>
 </tr>
 <tr>
 <td>Artificial cerebrospinal fluid</td>
 <td>30 ml</td>
 </tr>
 <tr>
 <td>Bovine pancreas trypsin</td>
 <td>40 mg</td>
 </tr>
 <tr>
 <td>Sheep testes hyaluronidase</td>
 <td>22.8 mg</td>
 </tr>
 <tr>
 <td>Kynurenic acid</td>
 <td>5 mg</td>
 </tr>
 </tbody>
</table>
Trypsin Solution
<table border="1">
 <tbody>
 <tr>
 <td>Item</td>
 <td>Volume or Mass</td>
 </tr>
 <tr>
 <td>SFM</td>
 <td>15 ml</td>
 </tr>
 <tr>
 <td>Ovomucoid trypsin inhibitor</td>
 <td>10 mg</td>
 </tr>
 </tbody>
</table>
Trypsin Inhibitor Solution
<table border="1">
 <tbody>
 <tr>
 <td>Item</td>
 <td>Volume</td>
 </tr>
 <tr>
 <td>Autoclaved water</td>
 <td>37 ml</td>
 </tr>
 <tr>
 <td>10X DMEM/F12</td>
 <td>10 ml</td>
 </tr>
 <tr>
 <td>30% Glucose</td>
 <td>2 ml</td>
 </tr>
 <tr>
 <td>7.5% NaHCO3</td>
 <td>1.5 ml</td>
 </tr>
 <tr>
 <td>1M HEPES</td>
 <td>0.5 ml</td>
 </tr>
 <tr>
 <td>Transferrin, Putrescine solution</td>
 <td>4 ml</td>
 </tr>
 <tr>
 <td>25 mg insulin solution</td>
 <td>4 ml</td>
 </tr>
 <tr>
 <td>Selenium</td>
 <td>100 &mu;l</td>
 </tr>
 <tr>
 <td>Progesterone</td>
 <td>100 &mu;l</td>
 </tr>
 </tbody>
</table>
Hormone Mix (100 ml total, store at -20 &deg;C)
<table border="1">
 <tbody>
 <tr>
 <td>Item</td>
 <td>Volume</td>
 </tr>
 <tr>
 <td>Autoclaved water</td>
 <td>37.5 ml</td>
 </tr>
 <tr>
 <td>10X DMEM/F12 (3:1)</td>
 <td>5 ml</td>
 </tr>
 <tr>
 <td>30% Glucose</td>
 <td>1 ml</td>
 </tr>
 <tr>
 <td>7.5% NaHCO3</td>
 <td>0.75 ml</td>
 </tr>
 <tr>
 <td>1M HEPES</td>
 <td>0.25 ml</td>
 </tr>
 <tr>
 <td>Hormone mix</td>
 <td>5 ml</td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>0.5 ml</td>
 </tr>
 <tr>
 <td>Penicillin-streptomycin</td>
 <td>0.5 ml</td>
 </tr>
 </tbody>
</table>
Serum Free Media EFH-SFM: add 10 &mu;l of EGF, 10 &mu;l of FGF, and 3.66 &mu;l of Heparin FBS-SFM: add 0.5 ml FBS
<table border="1">
 <tbody>
 <tr>
 <td>Item</td>
 <td>Volume</td>
 </tr>
 <tr>
 <td>Matrigel</td>
 <td>150 &mu;l</td>
 </tr>
 <tr>
 <td>SFM</td>
 <td>3.6 ml</td>
 </tr>
 </tbody>
</table>
Matrigel Solution Matrigel aliquot should be placed in a box of ice and allowed to thaw slowly over 4-5 hours to form a viscous liquid before mixing with SFM. This will ensure the formation of a smooth layer of Matrigel substrate. If not thawed slowly, the resulting substrate will contain clumps of Matrigel, possibly hindering cell migration.
<table border="1">
 <tbody>
 <tr>
 <td>Item</td>
 <td>Volume or Mass</td>
 </tr>
 <tr>
 <td>UltraPure Agarose</td>
 <td>300 mg in 10 ml ddH20</td>
 </tr>
 <tr>
 <td>SFM Heat Inactivated FBS</td>
 <td>8 ml 2 ml</td>
 </tr>
 </tbody>
</table>
Matrigel Solution Mix 8 ml of SFM with 2 ml heat inactivated FBS in a 15 cc falcon tube. Mix agarose with 10 ml ddH20 in an Erlenmeyer flask, and heat in a microwave for 30 sec in 10-sec intervals, ensuring to remove the solution from the microwave after each 10-sec interval and thoroughly mix. Following the final 10-sec microwave period, mix the agarose solution with the SFM/FBS solution and store in a 57 &deg;C water bath.
</td>
 </tr>
 </tbody>
</table>

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.

Kinematic analysis reveals that in the presence of a 250 mV/mm dcEF, undifferentiated NPCs exhibit highly directed and rapid galvanotaxis toward the cathode (Figure 5A, Movie 1). In the absence of a dcEF, random movement of the cells is observed (Figure 5B, Movie 2). At this field strength, &gt; 98% of undifferentiated NPCs migrate for the entire 6-8 hr for which they are imaged, and since dead cells do not migrate this suggests that they remain viable during this period in the abs...

Watch this Scientific Journal Video about A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field at JoVE.com

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

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 ...

Research

JoVE Journal

Neuroscience

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Electrode Positioning and Montage in Transcranial Direct Current Stimulation

Transcranial direct current stimulation (tDCS) is a technique that has been intensively investigated in the past decade as this method offers a ...

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 ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

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. ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

Remotely Supervised Transcranial Direct Current Stimulation: An Update on Safety and Tolerability

The remotely supervised tDCS (RS-tDCS) protocol enables participation from home through guided and monitored self-administration of tDCS treatment while ...