Name: using the dot assay to analyze migration of cell sheets
Uploaded: 2017-12-05T13:00:00
Description: Watch this Scientific Journal Video about Using the Dot Assay to Analyze Migration of Cell Sheets at JoVE.com

Forfatteren ønsker at takke Bhagawat Subramanian, Yi (Jason) Chang, Paul Randazzo og Carole Parent for læsning og kommentering på manuskriptet. Dette arbejde blev støttet af den murene Research Program af National Cancer Institute, National Institutes of Health.

1. belægning af retter (dag 1)
Bemærk: Sørg for ikke at efterlade fingeraftryk eller snavs i bunden af pladen, når du håndterer det.
<ol><li>Tø mus kollagen IV på isen, og fortynd det med 50 mM HCl (pH 1.3) for at forberede 3 mL af en 10 µg/mL kollagen IV løsning. Bemærk: Kollagen vil bundfald ved 37 ° C. Det er derfor vigtigt at holde kollagen løsning ved en lav temperatur mens optøning og arbejder med det. Undgå gentagne fryse-tø cykler ved at udarbejde passende størrelse delprøver og gemme...

<ol>
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J., 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=Malignant+MCF10CA1+cell+lines+derived+from+premalignant+human+breast+epithelial+MCF10AT+cells.">Malignant MCF10CA1 cell lines derived from premalignant human breast epithelial MCF10AT cells.</a> Breast Cancer Research and Treatment. 65 (2), 101-110 (2001).</li><li>Tang, B., Vu, M., 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=TGF-beta+switches+from+tumor+suppressor+to+prometastatic+factor+in+a+model+of+breast+cancer+progression.">TGF-beta switches from tumor suppressor to prometastatic factor in a model of breast cancer progression.</a> J Clin Invest. 112 (7), 1116-1124 (2003).</li><li>Lee, R. M., Stuelten, C. H., Parent, C. 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Under tumor progression celler vandrer fra væv af oprindelse til at invadere det omgivende væv og metastaserer til fjerne steder15. Migration af celler fra en celle koloni kan observeres i dot assay. Her, er dot assay illustreret ved at analysere den vandrende Fænotypen af humane brystkræftceller svar på EGF.
For at opnå nøjagtige og reproducerbare resultater flere trin i etableringen af dot er assays kritisk. Først, overfladebehandling af pladen bestemmer, om o...

Migration assays er almindeligt anvendt til at evaluere den invasive og metastatisk potentiale af tumor celler i vitro. Mest almindeligt, bruges sår eller Skrab analysen til at vurdere migration af epitel ark ind i en celle markeret område1,2,3. For at udføre de scratch assay, celler er belagt i en éncellelag og en "scratch" eller celle-området er lavet med en pipette spids. Scratch analysen er nem at sætte med almindeligt tilgængelige vævskultur forsyninger og kan udføres i flere godt plader, giver mulighed for behandling af flere prøver. Men som scratch celler er fysisk fjernet fra éncellelag og ofte undergår celledød. Derudover er ekstracellulære matrix knyttet til pladen ofte beskadiget under en aflysning proces. Ligeledes brugen af silicium indsætter (f.eks Ibidi kamre4) eller af stencils5,6,7 kan føre til mekanisk afbrydelse af cellerne og delvis fjernelse af matrix proteiner anvendes til overfladebehandling af plader. En anden ulempe ved assays overvågning lukning af sår eller rifter er deres begrænsede tidsforløb, som celle migration kan kun analyseres, indtil bunden er lukket.
Ved udførelsen af dot assay, er celler forgyldt som en cirkulær koloni på en overtrukket eller Ubestrøget plade8. Begrundelsen for denne plating strategi er at opnå celle ark med definerede kanter, der kan overføre eller invadere i de omkringliggende områder, celle-fri uden at forstyrre kulturen af fjernelse af celler eller skær. Det overordnede mål med dot assay er at observere migration af celle ark som målt ved kanten forskydning eller koloni diameter, samt at udføre time-lapse imaging for at analysere den vandrende Fænotypen af celler i højere spatio-temporal opløsning.
Celle migration kan blive påvirket af en række microenvironmental stikord som kemokiner, cytokiner og vækstfaktorer som EGF. EGF er en vækstfaktor, der udøver sin biologiske virkning via binding til dens receptor, EGF receptoren9, og øger invasive og metastatisk opførsel af tumor celler4,9,10. Her, dot analysen bruges til at studere EGF stimuleret celle migration i en menneskelig invasive, lunge kolonidannende bryst kræft celle linje (MCF10CA1a)8,11,12.

<table style="border-collapse:collapse;width:659pt" width="878">
	<colgroup>
		<col style="width:302pt" width="402" />
		<col style="width:160pt" width="213" />
		<col style="width:131pt" width="175" />
		<col style="width:66pt" width="88" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="width:302pt;height:15.75pt" width="402">MCF10CA1a cells</td>
			<td style="width:160pt" width="213">Karmanos Research Institute</td>
			<td class="xl18" style="width:131pt" width="175">N/A</td>
			<td style="width:66pt" width="88">cells used for asay</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">mouse collagen IV</td>
			<td>BD Biosciences</td>
			<td class="xl18">354233</td>
			<td>used to coat plates</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">trypsin / EDTA</td>
			<td>Thermo Fisher</td>
			<td class="xl19">25300054</td>
			<td>used to remove cells from tissue culture plates</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">DPBS&#160;</td>
			<td>Mediatech</td>
			<td class="xl18">21-031-CV</td>
			<td>used to wash cells</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">DMEM / F12</td>
			<td>Thermo Fisher</td>
			<td class="xl18">11320082</td>
			<td>used as culture medium</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">horse serum</td>
			<td>Thermo Fisher</td>
			<td class="xl18">26050088</td>
			<td>used to supplement culture medium</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">12well glass bottom plate</td>
			<td>MatTek</td>
			<td class="xl18">P12G-1.5-14-F</td>
			<td>used to grow cells for imaging</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">LPA (1-Oleoyl Lysophosphatidic Acid )</td>
			<td>Cayman</td>
			<td class="xl18">10093</td>
			<td>used to stimulate cells</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">EGF (epidermal growth factor, human recombinant)</td>
			<td>Peprotech</td>
			<td class="xl18">AF-100-15</td>
			<td>used to stimulate cells</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">fatty acid free BSA</td>
			<td>Sigma</td>
			<td class="xl18">A8806-1G</td>
			<td>used to make buffer for LPA and EGF</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">hematoxylin</td>
			<td>Sigma</td>
			<td class="xl18">HHS32</td>
			<td>used to stain colonies</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">eosin</td>
			<td>Electron Microscopy Sciences</td>
			<td class="xl18">26051-10</td>
			<td>used to stain colonies</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">37C, 5% CO2 incubator</td>
			<td>Sanyo</td>
			<td class="xl18">model MCO-18AIC (UV)</td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Zeiss AxioObserver with incubator chamber and motorized stage</td>
			<td>Zeiss</td>
			<td class="xl18">model Axio Observer.Z1</td>
			<td>used for time lapse imaging</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">ORCA Flash LT sCMOS camera</td>
			<td>BioVision</td>
			<td class="xl21">C11440-42U-KIT</td>
			<td>used for timelapse imaging in combination with Zeiss AxioObserver</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Metamorph</td>
			<td>BioVision</td>
			<td class="xl18">MMACQMIC</td>
			<td>software used for time lapse imaging</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">inverted microscope</td>
			<td>Olympus</td>
			<td class="xl18">model IX70</td>
			<td>used to count cells and to check colonies in tissue culture</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">plastic box</td>
			<td>Lock&#38;Lock</td>
			<td class="xl18">N/A</td>
			<td>used as humid chamber (any tight closing plastic box that fits the plates can be used)</td>
		</tr>
	</tbody>
</table>

using the dot assay to analyze migration of cell sheets

Selv om komplekse organismer vises statisk, er deres væv under en løbende omsætning. Som celler alder, flytte die, og er erstattet af nye celler, celler inden for væv i et stramt orkestreret måde. Under tumor udvikling, denne balance forstyrres, og tumorceller forlade epitel af oprindelse til at invadere den lokale mikromiljø, til at rejse til fjerne steder og i sidste ende udgør metastatisk tumorer på fjerne steder. Dot-analysen er en enkel, to-dimensionelle uhindret migration assay, til at vurdere den netto bevægelse celle ark ind i en celle-området og analysere parametre i celle migration ved hjælp af time-lapse billeddannelse. Her, dot assay er påvist ved hjælp af en human invasive, lunge kolonidannende breast cancer cellelinie, MCF10CA1a, til at analysere de celler vandrende svar til epidermal vækst faktor (EGF), som er kendt for at øge malignt potentiale af brystkræftceller og at ændre den vandrende Fænotypen af celler.

Dot analysen præsenteres her (figur 1) blev udført ved hjælp af invasive, lunge kolonidannende brystkræftceller (MCF10CA1a) som et modelsystem. Dot assay udbytter meget reproducerbare celle prikker selv i hånden af begyndere, hvilket gør det en praktisk og let at udføre analysen (fig. 1D). Dot assay kombineret med han farvning eller time-lapse imaging og efterfølgende partikel billede Velocimetri (PIV) gi...

Watch this Scientific Journal Video about Using the Dot Assay to Analyze Migration of Cell Sheets at JoVE.com

Using the Dot Assay to Analyze Migration of Cell Sheets

Ved hjælp af Dot analyse til at analysere Migration af celle ark

Celle migration er afgørende for udvikling, væv vedligeholdelse og reparation og tumordannelse, og er reguleret af vækstfaktorer, kemokiner og cytokiner. Denne protokol beskriver dot assay, en to-dimensionelle, uhindret migration assay at vurdere den vandrende Fænotypen af vedlagte, sammenhængende celle ark som svar på microenvironmental stikord.

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move ...

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