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...

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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. 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No conflicts of interest declared.

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>
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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 ...

a-galvanotaxis-assay-for-analysis-neural-precursor-cell-migration

Research

JoVE Journal

Neuroscience

12.4K Görüntüleme.  University of Toronto. Bu protokolü biz galvanotaksis sırasında nöral prekürsör hücre translokasyon edilen erişkin beyninin time-lapse görüntüleme sağlamak için bir doğru akım elektrik alanın uygulanmasına izin özel odaları oluşturmak nasıl gösterilmektedir.