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

Dette arbeidet er finansiert av Natural Sciences and Engineering Research Council of Canada (tilskudd # 249669 og # 482986) og Heart og Stroke Foundation of Canada (tilskudd # 485508). Forfatterne takker Youssef El-Hayek og Dr. Qi Wan for deres hjelp i utviklingen av eksperimentelle protokoller.

Alle prosedyrer som involverer dyr håndtering ble godkjent av Universitetet i Toronto Animal Care komiteen i samsvar med institusjonelle retningslinjer (protokoll nr. 20009387). Følgende metoder bør utføres ved hjelp av sterile verktøy og teknikker, i en laminær hette der det er aktuelt. I protokollen teksten nedenfor, refererer uttrykket &quot;EFH-SFM&quot; til serumfritt medier supplert med epidermal vekstfaktor, grunnleggende fibroblast vekstfaktor og heparin. EFH-SFM brukes når et...

<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>

Denne protokollen har blitt tilpasset fra de veletablerte metoder for tidligere studier 7-9. Galvanotactic kamre kan konstrueres ved hjelp av en rekke forskjellige teknikker, herunder konstruksjonen av en separat glass godt for innesperring av celle såing, eller ved hjelp av CO 2 laser ablasjon for microfabrication av den sentrale renne 10,11. Noen teknikker kan være mer arbeidskrevende eller kostbare enn andre. Vi har beskrevet en enkel og kostnadseffektiv analyse for å konstruere en...

<table border="1"><tbody><tr><td height="44"> Navn på element </td><td> Selskapet </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td></td><td></td><td></td><td> Neural Precursor celleisolasjon </td></tr><tr><td> 2M NaCl </td><td> Sigma </td><td> S5886 </td><td> 11.688 g oppløses i 100 ml dH 2 O </td></tr><tr><td> 1M KCl </td><td> Sigma </td><td> P5405 </td><td> 7.456 g oppløses i 100 ml dH 2 O </td></tr><tr><td> 1M MgCl 2 </td><td> Sigma </td><td> M2393 </td><td> 20.33 g oppløses i 100 ml dH 2 O </td></tr><tr><td> 155 mm NaHCO 3 </td><td> Sigma </td><td> S5761 </td><td> 1.302 g oppløses i 100 ml dH 2 O </td></tr><tr><td> 0.5M Glukose </td><td> Sigma </td><td> G6152 </td><td> 9,01 g oppløst i 100ml dH 2 O </td></tr><tr><td> 108 mm CaCl 2 </td><td> Sigma </td><td> C7902 </td><td> 1,59 g oppløses i 100 ml dH 2 O </td></tr><tr><td> Penicillin-streptomycin </td><td> Gibco </td><td> 15070 </td><td></td></tr><tr><td> Bovine bukspyttkjertelen trypsin </td><td> Sigma </td><td> T1005 </td><td></td></tr><tr><td> Sauer testiklene hyaluronidase </td><td> Sigma </td><td> H6254 </td><td></td></tr><tr><td> Kynurenic syre </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% Glukose </td><td> Sigma </td><td> G6152 </td><td></td></tr><tr><td> 7,5% NaHCO 3 </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 oppløses i 100 ml dH 2 O </td></tr><tr><td> L-glutamin </td><td> Gibco </td><td> 25030 </td><td></td></tr><tr><td> EGF </td><td> Invitrogen </td><td> PMG8041 </td><td> Rekonstitueres i 1 ml av hormon mix og alikvot inn 20 ul enheter. </td></tr><tr><td> FGF </td><td> Invitrogen </td><td> PHG0226 </td><td> Rekonstitueres i 0,5 ml av hormon mix og alikvot inn 20 ul enheter. </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 oppløses i 4 ml dH 2 0 </td></tr><tr><td> Putrescine </td><td> Sigma </td><td> P7505 </td><td> Oppløse 9,61 mg til Apo-transferrin sårensning </td></tr><tr><td> Insulin </td><td> Sigma </td><td> I5500 </td><td> Oppløs 25 mg inn 0,5 ml 0,1 N HCl og legge til 3,5 ml dH 2 0 </td></tr><tr><td> Selen </td><td> Sigma </td><td> S9133 </td><td></td></tr><tr><td> Progesteron </td><td> Sigma </td><td> P6149 </td><td></td></tr><tr><td> Standard Disseksjonsverktøyer </td><td> Fin Science Verktøy </td><td></td><td></td></tr><tr><td> Disseksjon mikroskop </td><td> Zeiss </td><td> STEMI 2000 </td><td></td></tr><tr><td></td><td></td><td></td><td> Galvanotaxis Chamber Forberedelse </td></tr><tr><td> Square glassdeksel lysbilder </td><td> VWR </td><td> 16004 </td><td></td></tr><tr><td> 6N saltsyre </td><td> VWR </td><td> BDH3204-1 </td><td></td></tr><tr><td> Høyvakuum fett </td><td> Dow Corning </td><td></td><td></td></tr><tr><td> 60 mm petriskåler </td><td> Fisher Scientific </td><td> 0875713A </td><td></td></tr><tr><td> Poly-L-lysin </td><td> Sigma </td><td> P4707 </td><td></td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354234 </td><td> Tine og alikvot inn 150 pl enheter </td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 10082139 </td><td> Bare bruke hvis indusere NPC differensiering, ellers bruker SFM + EFH kultur media som angitt ovenfor </td></tr><tr><td> Counting mikroskop </td><td> Olympus </td><td> CKX41 </td><td></td></tr><tr><td></td><td></td><td></td><td> Lever Cell Time-Lapse Imaging </td></tr><tr><td> Sølv 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 Inactivrende FBS </td><td> Sigma </td><td> 16140071 </td><td></td></tr><tr><td> PVC rør </td><td> Fisher Scientific </td><td> 80000006 </td><td> 3/32 &quot;ID x 5/32&quot; OD </td></tr><tr><td> Blekemiddel </td><td> Clorox </td><td></td><td></td></tr><tr><td> 10 cc sprøyte </td><td> BD </td><td> 309604 </td><td></td></tr><tr><td> 18 gauge nål </td><td> BD </td><td> 305195 </td><td></td></tr><tr><td> Dremel drill </td><td> Dremel </td><td> Modell 750 </td><td></td></tr><tr><td> Invertert mikroskop utstyrt med fuktet, inkubert kammer </td><td> Zeiss </td><td> Axiovert-200M </td><td></td></tr><tr><td></td><td></td><td></td><td> Oppskrifter <table border="1"><tbody><tr><td> Element </td><td> Volum </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 MgCl 2 </td><td> 0,32 ml </td></tr><tr><td> 155mm NaHCO 3 </td><td> 16,9 ml </td></tr><tr><td> 1M Glukose </td><td> 1 ml </td></tr><tr><td> 108 mm CaCl 2 </td><td> 0,09256 ml </td></tr><tr><td> Penicillin-streptomycin </td><td> 1 ml </td></tr><tr><td> Autoklaverte vann </td><td> 74 ml </td></tr></tbody></table> Kunstig cerebrospinalvæsken <table border="1"><tbody><tr><td> Element </td><td> Volum eller Mass </td></tr><tr><td> Kunstig cerebrospinalvæsken </td><td> 30 ml </td></tr><tr><td> Bovine bukspyttkjertelen trypsin </td><td> 40 mg </td></tr><tr><td> Sauer testiklene hyaluronidase </td><td> 22,8 mg </td></tr><tr><td> Kynurenic syre </td><td> 5 mg </td></tr></tbody></table> &lt;strong&gt; Trypsin Solution <table border="1"><tbody><tr><td> Element </td><td> Volum eller 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> Element </td><td> Volum </td></tr><tr><td> Autoklaverte vann </td><td> 37 ml </td></tr><tr><td> 10X DMEM/F12 </td><td> 10 ml </td></tr><tr><td> 30% Glukose </td><td> 2 ml </td></tr><tr><td> 7,5% NaHCO 3 </td><td> 1,5 ml </td></tr><tr><td> 1M HEPES </td><td> 0,5 ml </td></tr><tr><td> Transferrin, putrescine løsning </td><td> 4 ml </td></tr><tr><td> 25 mg insulin løsning </td><td> 4 ml </td></tr><tr><td> Selen </td><td> 100 &amp; mu, L </td></tr><tr><td> Progesteron </td><td> 100 ul </td></tr></tbody></table> Hormon Mix (100 ml totalt, lagre ved -20 ° C) <table border="1"><tbody><tr><td> Element </td><td> Volum </td></tr><tr><td> Autoklaverte vann </td><td> 37,5 ml </td></tr><tr><td> 10X DMEM/F12 (3:1) </td><td> 5 ml </td></tr><tr><td> 30% Glukose </td><td> 1 ml </td></tr><tr><td> 7,5% NaHCO 3 </td><td> 0,75 ml </td></tr><tr><td> 1M HEPES </td><td> 0,25 ml </td></tr><tr><td> Hormon mix </td><td> 5 ml </td></tr><tr><td> L-glutamin </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: legg 10 pl EGF, 10 pl FGF, og 3,66 ul av heparin FBS-SFM: tilsett 0,5 ml FBS <taBLE border = &quot;1&quot;&gt; <tbody><tr><td> Element </td><td> Volum </td></tr><tr><td> Matrigel </td><td> 150 pl </td></tr><tr><td> SFM </td><td> 3,6 ml </td></tr></tbody></table> Matrigel Solution Matrigel alikvot bør plasseres i en boks av is og tillatt å tine langsomt over 4-5 timer for å danne en viskøs væske før blanding med SFM. Dette vil sikre at det dannes et jevnt lag av Matrigel substrat. Hvis ikke Tint langsomt, vil den resulterende substratet inneholde klumper av Matrigel, muligens hindering cellemigrering. <table border="1"><tbody><tr><td> Element </td><td> Volum eller Mass </td></tr><tr><td> Ultrapure Agarose </td><td> 300 mg i 10 ml DDH 2 0 </td></tr><tr><td> SFM Varmeinaktivert FBS </td><td> 8 ml 2 ml </td></tr></tbody></table> MatrigelLøsning Bland 8 ml SFM med 2 ml varmeinaktivert FBS i en 15 cc falk tube. Mix agarose med 10 ml DDH 2 0 i en Erlenmeyer kolbe, og varme i en mikrobølgeovn i 30 sekunder i 10-sek intervaller, sørge for å fjerne løsningen fra mikrobølgeovn etter hver 10-sek intervall og grundig blande. Etter den siste 10-sek mikrobølgeovn periode, bland agarose løsningen med SFM / FBS løsning og oppbevares på et 57 ° C vannbad. </td></tr></tbody></table>

a galvanotaxis assay for analysis of neural precursor cell migration kinetics in an externally applied direct current electric field

Oppdagelsen av nevrale stilk og progenitor celler (betegnet med samlebetegnelsen neural forløper celler) (NPC) i den voksne hjernen hos pattedyr har ført til en kropp av forskning rettet mot å utnytte de multipotent og proliferativ egenskapene til disse cellene for utvikling av neuroregenerative strategier. En kritisk steg for å lykkes med slike strategier er mobilisering av NPCer mot en lesjon nettstedet etter eksogene transplantasjon eller for å forbedre responsen til de endogene forløperne som er funnet i den periventrikulær regionen i CNS. Følgelig er det viktig å forstå hvilke mekanismer som fremmer, veilede og forbedre NPC migrasjon. Vårt arbeid fokuserer på utnyttelse av likestrøm elektriske felt (dcEFs) for å fremme og direkte NPC migrasjon - et fenomen kjent som galvanotaxis. Endogene fysiologiske elektriske felt fungere som kritiske signaler for celle migrasjon under normal utvikling og såret reparasjon. Farmakologisk forstyrrelse avtrans-nevralrøret potensial i Axolotl embryoer forårsaker alvorlige utviklingsforstyrrelser misdannelser en. I sammenheng med sårtilheling, er frekvensen av reparasjon av sårede hornhinnen direkte korrelert med størrelsen av epiteliale såret potensialet som oppstår etter skaden, som vist ved farmakologisk forsterkning eller avbrudd av denne DCEF 2-3. Vi har vist at voksne subependymal NPCer gjennomgå rask og regissert katodisk migrasjon in vitro når de utsettes for en eksternt påført DCEF. I denne protokollen beskriver vi vår lab teknikker for å skape en enkel og effektiv galvanotaxis analysen for høy oppløsning, langsiktige observasjon av rettet cellen kroppen translokasjon (migrasjon) på en encellet nivå. Denne analysen vil være egnet for å undersøke mekanismer som regulerer DCEF transduksjon i cellulært motilitet gjennom bruk av transgene eller knockout mus, korte interfererende RNA, eller bestemte reseptor agonister / antagonister.

Kinematisk analyse viser at i nærvær av en 250 mV / mm DCEF, udifferensierte NPCer oppviser sterkt rettet og raske galvanotaxis mot katoden (figur 5A, Film 1). I fravær av en DCEF, er tilfeldig bevegelse av cellene observert (figur 5B, Film 2). På dette feltstyrke,&gt; 98% av udifferensierte NPCer migrerer for hele 6-8 hr som de er avbildet, og siden døde celler ikke migrere dette tyder på at de forblir levedyktig i denne perioden i fravær eller nærvær av en DCEF. <p class=...

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

I denne protokollen viser vi hvordan du kan lage tilpassede kammer som tillater bruk av en likestrøm elektrisk felt for å aktivere time-lapse avbildning av voksen hjerne avledet nevrale forløper celle translokasjon under galvanotaxis.

En Galvanotaxis Assay for analyse av Neural forløper celle migrasjon Kinetics i en eksternt anvendes Direct Current elektrisk felt

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

Reproduserbar Mouse hoftenervene Crush og påfølgende vurdering av Regeneration av 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

Analyse av Neural Crest Migrasjon og differensiering av Cross-arter 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

Den Neuroblast analysen: en analyse for generering og berikelse av neuronal stamceller fra Skille Neural Stem Cell familie Bruke flowcytometrisystemer

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

Isolering og kultur neural crest Celler fra Embryonic murin 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

En kvantitativ Cell Migration analysen for Murine resistente Neural stamceller

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

Langsiktig Behavioral Sporing av frittsvømmende 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

Eksternt veiledet Transkraniell direkte gjeldende stimulering: En oppdatering på sikkerhet og toleranse

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