Name: a device for performing cell migration/wound healing in a 96-well plate
Uploaded: 2017-03-07T15:00:00
Description: Watch this Scientific Journal Video about A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate at JoVE.com

Forfatterne vil gerne takke Mr. Tam Po Leung, Mr. Wong Chi Kin og tekniske personale på Science Faculty Workshop, Hongkong Baptist University, for deres tekniske færdigheder og rådgivning i at gøre prototypen på denne wounder.

1. Introduktion til Dele af wounder (figur 1) <ol><li> Forbered holder stiften ved fastsættelse af sårende stifter i samme afstand. Hold pipettespidserne og justere spidsen højden ved at flytte de justerbare såring stifter op og ned. </li><li> Monter indstillelige føring-bar på forskellige mærker af 96-brønds kulturplader og sikre, at såret område er i en fast position. </li><li> Fastgør den justerbare såret pin ved at stramme den sekskantede skrue. Fix det ledende-bar på plads ved at stramme sekskant hæ...

<ol>
	<li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-cell+invasion+and+migration:+diversity+and+escape+mechanisms.">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nat Rev Cancer. 3 (5), 362-374 (2003).</li><li>Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prespecification+and+plasticity:+shifting+mechanisms+of+cell+migration.">Prespecification and plasticity: shifting mechanisms of cell migration.</a> Curr Opin Cell Biol. 16 (1), 14-23 (2004).</li><li>Lampugnani, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+into+a+wounded+area+in+vitro.">Cell migration into a wounded area in vitro.</a> Methods Mol Biol. 96, 177-182 (1999).</li><li>Sholley, M. M., Gimbrone, M. A., Cotran, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+migration+and+replication+in+endothelial+regeneration:+a+study+using+irradiated+endothelial+cultures.">Cellular migration and replication in endothelial regeneration: a study using irradiated endothelial cultures.</a> Lab Invest. 36 (1), 18-25 (1977).</li><li>Gotlieb, A. I., Spector, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+into+an+in+vitro+experimental+wound:+a+comparison+of+porcine+aortic+endothelial+and+smooth+muscle+cells+and+the+effect+of+culture+irradiation.">Migration into an in vitro experimental wound: a comparison of porcine aortic endothelial and smooth muscle cells and the effect of culture irradiation.</a> Am J Pathol. 103 (2), 271-282 (1981).</li><li>Chen, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+migration+chip+for+chemotaxis-based+microfluidic+selection+of+heterogeneous+cell+populations.">Single-cell migration chip for chemotaxis-based microfluidic selection of heterogeneous cell populations.</a> Sci Rep. 18 (5), 9980 (2015).</li><li>Yarrow, J. C., Periman, Z. E., Westwood, N. J., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+cell+migration+assay+using+scratch+wound+healing,+a+comparsion+of+image-based+readout+methods.">A high-throughput cell migration assay using scratch wound healing, a comparsion of image-based readout methods.</a> BMC Biotechnol. 4, 21 (2004).</li><li>Lauder, H., Frost, E. E., Hiley, C. R., Fan, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+repair+process+involved+in+the+repair+of+a+cell+monolayer+using+an+in+vitro+model+of+mechanical+injury.">Quantification of the repair process involved in the repair of a cell monolayer using an in vitro model of mechanical injury.</a> Angiogenesis. 2 (1), 67-80 (1998).</li><li>Yue, P. Y. K., Leung, E. P. Y., Mak, N. K., Wong, R. N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+method+for+quantifying+cell+migration/+wound+healing+in+96-well+plates.">A simplified method for quantifying cell migration/ wound healing in 96-well plates.</a> J. Biomol Screen. 15 (4), 427-433 (2010).</li><li>Yue, P. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidation+of+the+mechanisms+underlying+the+angiogenic+effects+of+ginsenoside+Rg(1)+in+vivo+and+in+vitro.">Elucidation of the mechanisms underlying the angiogenic effects of ginsenoside Rg(1) in vivo and in vitro.</a> Angiogenesis. 8 (3), 205-216 (2005).</li><li>Kwok, H. H., Chan, L. S., Poon, P. Y., Yue, P. Y., Wong, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ginsenoside-Rg1+induces+angiogenesis+by+the+inverse+regulation+of+MET+tyrosine+kinase+receptor+expression+through+miR-23a.">Ginsenoside-Rg1 induces angiogenesis by the inverse regulation of MET tyrosine kinase receptor expression through miR-23a.</a> Toxicol Appl Pharmacol. 287 (3), 276-283 (2015).</li></ol>

Vores celle wounder har flere unikke funktioner i at løse problemerne med traditionelle celle migration assays. Den 8-kanals mekanisk celle wounder er lavet af høj kvalitet rustfrit stål (stål 304) med en lang levetid, der kan steriliseres ved autoklavering. Næsten alle kommercielle mærker af 96-brønds dyrkningsplader findes på markedet, kan anvendes med denne mekaniske celle wounder grund af den indstillelige styrestangen. Udformningen af ​​den justerbare styrestangen sikrer også, at skrabe område er i ce...

Cellemigrering spiller en vigtig rolle i cellulære processer, såsom embryogenese, neurogenese, angiogenese, sårheling, mucosal reparation, epitel--mesenchymal-overgange under normal udvikling og sygdomssituationen 1. Det er en kompliceret proces, der kræver det en samordning af talrige inter- og intra-cellulære begivenheder, herunder signalmolekyle interaktioner, celle polarisering, cytoskeletal reorganisering, matrix remodeling, membran fremspring og dynamiske celle-celle adhæsion modulation 2. Ved at studere celle migration, kan bestemmes opdagelsen og validering af kemikalier eller biomolekyler, der fører til celle bevægelse og dens relaterede biokemiske veje. Denne grundlæggende proces kan bruges med fordel som en proxy for forskellige applikationer, såsom sygdom behandling og narkotika målretning. Den sårede analysen er en af celle migration assays 3. Her, enindividuel prøve af celler vil blive delvist fjernes mekanisk for at frembringe en blottede område, hvor celler vil migrere til at dække området. Procentdelen af ​​genvinding på et bestemt tidspunkt vil blive overvåget. Ved håndtering stort antal prøver, kan emner som den eksperimentelle omkostninger og krydskontaminering inden prøverne være problematisk. Selvom mange kommercielle værktøjer og assays er tilgængelige til undersøgelse af cellemigrering 4, 5, 6, mange af dem krævede dyrt og sofistikeret udstyr og stadig behov for forbedringer. Af disse grunde er en 8-kanals mekanisk celle wounder (figur 1) udviklet. Vores 8-kanals mekanisk celle wounder har indarbejdet flere unikke funktioner i at løse de ovennævnte problemer. Det giver fleksibilitet til at udføre cellemigration / sårede assays i 96-brønds dyrkningsplade format. Den justerbare guiding bar design sikrer, at bunden område er i den centrale position i hver brønd. Desuden kan højden af ​​de ledende stænger justeres således at wounder er anvendelig til forskellige mærker af dyrkningsplader. Endelig den justerbare såret pin design muliggør en jævn kontakt pipettespidserne med kulturen pladens overflade for at opnå en samtidig og reproducerbar sårdannelse 7, 8.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well cell culture plate</td>
			<td>Nunc</td>
			<td>167008</td>
			<td>Other brands of 96-well cell culture plate can also be used</td>
		</tr>
		<tr>
			<td>P10 pipette tips</td>
			<td>Axygen</td>
			<td>301-03-051</td>
			<td>Short P10 pipette tip is more easy to create a clear wound</td>
		</tr>
		<tr>
			<td>Wounder</td>
			<td>R&#38;P Technology Limited</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Sigma</td>
			<td>M2520-1L</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>26140079</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
		<tr>
			<td>Heparin sodium salt from porcine intestinal mucosa</td>
			<td>Sigma</td>
			<td>H3393</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
		<tr>
			<td>Gelatin from bovine skin</td>
			<td>Sigma</td>
			<td>G9391</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
	</tbody>
</table>

a device for performing cell migration/wound healing in a 96-well plate

Den cellemigration / sårede assay er en almindeligt anvendt metode til at studere celle migration og andre biologiske processer, såsom angiogenese og tumormetastase. I dette assay dyrkes celler til dannelse af et sammenflydende monolag og en mekanisk sår er skabt ved at skrabe med en enhed. Derefter vandringshastigheden af ​​cellerne mod det blottede område kan overvåges ved billeddannelse. Vores 8-kanals mekanisk wounder er designet til at håndtere de fleste af de problemer, der er forbundet med cellemigrationsassay. For det første kan vores wounder let steriliseres ved autoklavering eller med almindelige desinfektionsmidler. For det andet tillader de enkelte justerbare ben selv kontakt med cellekultur pladen, så der kan skabes skarpe og reproducerbare sår. For det tredje, de ledende bjælker på begge sider af wounder sikre konsekvent sårdannelse position i hver brønd. Brugen af ​​engangs plast pipettespidser til sårede yderligere kan give bedre håndtering af wounder samt at minimere tværs SNAVSETion. Som konklusion kan vores celle wounder give forskerne et brugervenligt og reproducerbar indretning til udøvelse af cellemigrationsassay anvendelse af standard 96-brønds dyrkningsplade.

Denne 8-kanals mekaniske wounder er konstrueret til at ridse en celle monolag for at udføre den cellemigrationsassay. Det er en brugervenlig enhed, der kan udføre celle skrabe analyser i 96-brønds plader på mindre end et minut uden særlig uddannelse. Denne wounder kan introducere viklede områder på cellemonolag med en ensartet bredde på ca. 600 um og med skarpe kanter (5 og 6). Bredden af ​​sår afhænger af mærke af pipettespidser og bruger...

Watch this Scientific Journal Video about A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate at JoVE.com

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

Her præsenteres en protokol til at udføre en semi-high throughput cellemigrationsassay på en 96-brønds celledyrkningsplade. Denne protokol er en hurtig, enkel og økonomisk metode til at skabe ensartede scratch sår på en celle monolag.

En indretning til udførelse Cell Migration / Wound Healing i en plade med 96 brønde

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

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