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

Der Autor bedankt sich bei Bhagawat Subramanian, Chang Yi (Jason), Paul Randazzo und Carole Parent fürs Lesen und kommentieren auf dem Manuskript. Diese Arbeit wurde unterstützt durch Intramurale Research Program des National Cancer Institute, National Institutes of Health.

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<ol><li>Tauen Sie Maus Kollagen IV auf Eis auf, und verdünnen Sie es mit 50 mM HCl (pH 1.3) um 3 mL einer 10 µg/mL Kollagen IV Lösung vorzubereiten. Hinweis: Kollagen wird Niederschlag bei 37 ° C. Es ist daher wichtig, die Kollagen-Lösung bei niedriger Temperatur zu halten, beim Auftauen und mit ihm arbeiten. Vermeiden Sie wiederholte Frost-Tau-Wec...

<ol>
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L., HGF, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LPA,+HGF,+and+EGF+utilize+distinct+combinations+of+signaling+pathways+to+promote+migration+and+invasion+of+MDA-MB-231+breast+carcinoma+cells.">LPA, HGF, and EGF utilize distinct combinations of signaling pathways to promote migration and invasion of MDA-MB-231 breast carcinoma cells.</a> BMC Cancer. 13, 501 (2013).</li><li>Santner, S. J., Dawson, P. 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. A., Losert, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cell+migration+over+long+time+scales+reveals+distinct+phenotypes.">Collective cell migration over long time scales reveals distinct phenotypes.</a> Convergent science physical oncology. 2 (2), 025001 (2016).</li><li>Lee, R. M., Kelley, D. H., Nordstrom, K. N., Ouellette, N. T., Losert, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+stretching+and+rearrangement+in+epithelial+sheet+migration.">Quantifying stretching and rearrangement in epithelial sheet migration.</a> New J Phys. 15 (2), (2013).</li><li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144 (5), 646-674 (2011).</li><li>Grada, A., Otero-Vinas, M., Prieto-Castrillo, F., Obagi, Z., Falanga, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+techniques+made+simple:+analysis+of+collective+cell+migration+using+the+wound+healing+assay.">Research techniques made simple: analysis of collective cell migration using the wound healing assay.</a> J Invest Dermatol. 137 (2), 11-16 (2017).</li><li>Rosen, P., Misfeldt, D. 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Migrieren Sie bei Fortschreiten der Tumorzellen vom Gewebe des Ursprungs das umliegende Gewebe einzudringen und Metastasen in entfernten Standorten15. Wanderung von Zellen von einer Zelle Kolonie kann in der Dot-Probe beobachtet werden. Hier ist der Dot-Test dargestellt, durch die Analyse des wandernden Phänotyps von menschlichen Brustkrebszellen in Reaktion auf EGF.
Um genaue und reproduzierbare Ergebnisse zu erhalten mehrere Schritte bei der Einrichtung des Punktes s...

Migration-Assays sind weit verbreitet, die invasive und metastatische Potential der Tumor Zellen in Vitrozu bewerten. Am häufigsten wird die Wunde oder Kratzer Assay verwendet, um Migration von epithelialen Blätter in eine Zelle gelöscht Bereich1,2,3bewerten. Um der Scratch-Test durchzuführen, sind Zellen in einem monomolekularen Film beschichtet und einen "Kratzer" oder zellfreie Bereich wird mit einer Pipettenspitze erstellt. Der Scratch-Test ist einfach mit handelsüblichen Gewebekultur Lieferungen einzurichten und im Multi-well-Platten, so dass für die Verarbeitung mehrerer Proben durchgeführt werden kann. Jedoch da der Kratzer hergestellt wird, Zellen sind physisch aus der Monolage entfernt und oft durchlaufen Zelltod. Extrazellulärer Matrix an der Platte befestigt ist darüber hinaus oft kratzen dabei beschädigt. Ebenso die Verwendung von Silizium wird eingefügt (z. B. Ibidi Kammern4) oder Schablonen5,6,7 zu mechanischen Störungen der Zellen und der teilweisen Entfernung der Matrixproteine Beschichtung führen die Platten. Ein weiterer Nachteil von Assays Überwachung Verschluss von Wunden oder Kratzer ist ihre begrenzte Zeit natürlich da Zellwanderung nur analysiert werden kann, bis der Kratzer geschlossen wird.
Bei der Durchführung des Dot-Tests, sind Zellen als eine kreisförmige Kolonie auf einer beschichteten oder unbeschichteten Platte8vergoldet. Die Gründe für diese Beschichtung-Strategie soll Zelle Blätter mit definierten Kanten zu erhalten, die dringen in die Zelle-freie Umgebung ohne zu stören die Kultur durch Abbau von Zellen oder Beilagen oder migrieren können. Das übergeordnete Ziel des Dot-Assays ist Migration der Zelle Blätter gemessen am Rande Verschiebung oder Kolonie Durchmesser, sowie hinsichtlich durchführen, Time-Lapse imaging zu analysieren, die wandernden Phänotyp der Zellen in räumlich-zeitliche Auflösung beobachten.
Zellwanderung kann durch eine Vielzahl von microenvironmental Signale wie Chemokine, Zytokine und Wachstumsfaktoren wie EGF beeinträchtigt werden. EGF ist ein Wachstumsfaktor, der übt seine biologische Wirkung über Bindung an seinen Rezeptor EGF-Rezeptor-9, und invasive und metastasierten Verhalten von Tumor Zellen4,9,10erhöht. Hier ist der Dot-Test zur Untersuchung EGF stimuliert Zellwanderung in eine menschliche invasive, Lunge koloniebildenden Brust Krebs Zelle Linie (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

Auch wenn komplexe Organismen statisch erscheinen, sind ihre Gewebe unter einem kontinuierlichen Umsatz. Als Alter Zellen, bewegen sterben und werden durch neue Zellen ersetzt Zellen in den Geweben in gewissem Sinne dicht orchestrierte. Während der Entwicklung von Tumoren ist dieses Gleichgewicht gestört, und Tumorzellen lassen das Epithel des Ursprungs, der lokalen Mikroumgebung, ferne Orte bereisen und bilden schließlich metastasierten Tumoren an entfernten Standorten zu erobern. Die Dot-Assay ist ein einfache, zweidimensionale ungezwungene Migration Assay, Nettobewegung Zelle Blätter in einem zellfreien Bereich bewerten und Parameter der Zellwanderung mit Zeitraffer-Bildgebung zu analysieren. Hier der Dot-Test zeigt sich mit einem menschlichen invasive, Lunge koloniebildenden Brust-Krebs-Zell-Linie, MCF10CA1a, wandernden Reaktion der Zellen auf epidermalen Wachstumsfaktor (EGF), analysieren die bekannt ist, bösartige Potenzial von Brustkrebs-Zellen zu erhöhen und die wandernden Phänotyp der Zellen ändern.

Die Dot-Assay präsentiert hier (Abbildung 1) erfolgte mittels invasiver, Lunge koloniebildenden Brustkrebs-Zellen (MCF10CA1a) als Modellsystem. Der Dot-Test ergibt hoch reproduzierbare Zelle Punkte selbst in der Hand von Anfängern, so dass es eine bequeme und leicht auszuführende Assay (Abbildung 1D). Die Dot-Assay kombiniert mit er Färbung oder Zeitraffer Bildgebung und nachfolgenden Partikel Image Velocimet...

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

Mit Hilfe der Dot-Assay, Migration der Zelle Blätter zu analysieren

Zellmigration ist entscheidend für Entwicklung, Gewebe-Wartung und Reparatur und Tumorgenese und wird durch Zytokine, Wachstumsfaktoren und Chemokine reguliert. Dieses Protokoll beschreibt Dot-Test, eine zweidimensionale, ungezwungene Migration Assay den wandernden Phänotyp der beigefügten, geschlossene Zelle Blätter als Reaktion auf microenvironmental Hinweise zu beurteilen.

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

The overall goal of the dot assay is to assess the migratory phenotype of attached cohesive cell sheets in response to microenvironmental cues. This method can help answer key questions in the cancer field, such as how cytokines or growth factors alter tumor cell motility. The main advantage of the dot assay is that it is easily performed and used for reproducible results.Though this method can provide insight into cell motility, it also lends itself to a range of subsequent assays, such as staining of cells to assess growth colony morphology and cell scattering, immunostaining, protein and DNA or RNA analysis, or injection of cells into animals. To begin, thaw mouse collagen four on ice, and dilute it with 50 micromolars HCL to prepare three milliliters of a 10 microgram per milliliter collagen four solution. Add 250 microliters of collagen four solution to the wells of 12-well glass-bottom plates, and place them into an appropriately sized, tight-closing plastic box.Place wet paper towel over and around the plates to manufacture a humid chamber, and close the box. Then, incubate the plates overnight at room temperature. The next morning, to move non-absorbed collagen and buffer, use deionized water to rinse the plates twice, directing the water to the edge of the plate well, rather than the edge formed by the edge bottom and the cover slip.Air dry the plates in a laminar flow hood, and use them immediately or store them at four degrees Celsius for up to five days. To prepare cell suspension, use cells that have been grown in a 60 millimeter tissue culture dish in DMEMF12 medium, supplemented with 5%horse serum to 80%confluence. Use calcium-and magnesium-free DPBS to rinse the cells once, then add 500 microliters of room temperature trypsin EDTA, and incubate the cells at 37 degrees Celsius in 5%CO2 in a humidified atmosphere for three to five minutes.Suspend the detached cells in 4.5 milliliters of culture medium to stop trypsin activity, and use a hemocytometer to count a 20 microliter aliquot of the cell suspension. Pull out the remaining cells in a 50 milliliter conical tube by centrifugation at 200 times gravity for three minutes. Then, aspirate the supernatant, and re-suspend the cells in culture medium to three times 10 to the six cells per milliliter.To plate the cells at the center of each coverslip of the collagen four coated 12-well glass-bottom plate, place a 10 microliter drop of cell suspension, taking care not to touch or scratch the coating. Plating of a non-circular drop will not yield a circular cell sheet, and if the coating is touched or scratched, it will prevent the cells from adhering well. Incubate the plate at 37 degrees Celsius in a humidified atmosphere for 30 minutes, to allow the cells to attach.Then, check for attachment under an inverted microscope. With one milliliter of culture medium, gently wash the wells twice, to remove non-attached cells, then add one milliliter of culture medium to each well. Check under an inverted microscope that no floating cells remain, otherwise wash the wells again.Incubate the cells overnight at 37 degrees Celsius in 5%CO2 in a humidified atmosphere to obtain well-defined cell sheets. Using an inverted microscope, check all the wells to ensure that cell colonies have grown properly. If necessary, mark unsuitable wells and do not use them.Label each well of the plate appropriately for each condition investigated. Run each condition in duplicate or triplicate. Change the medium to starve the cells in DMEMF12 containing 0.1%horse serum, three hours before cells are stimulated.Then, stimulate the cells by adding EGF or other desired mediators. Use a one microliter pipetter, or swirl the plate gently to mix the medium. Incubate the plate for one to four days to observe edge displacement and edge contour.To carry out the H and E staining, wash the cells with PBS twice and fix them for approximately two minutes. Add hematoxylin for approximately two minutes to stain the nuclei. Wash the cells with tap water for five to 10 minutes, until the nuclei are blue.Then, stain the cytoplasm with eosin for two minutes before rinsing the cells with deionized water. Air dry the plate, then flip it over and use a ruler to measure the dot diameter. Alternatively, take pictures of the plate with a camera, and use ImageJ to analyze the dot diameter.To carry out time lapse microscopy, switch on the incubator microscope, and adjust the temperature to 37 degrees Celsius. Fill the humidifier with DH2O, then adjust the CO2 to 5%Ensure that the incubator chamber is humidified, and that the motorized stage can move freely. Then, close the incubator chamber and allow the microscope to adjust to 37 degrees Celsius.Place the plate on the stage of the incubator microscope and set up the stage list, then image two opposing edges and the center of each cell dot. Using a 10X objective, take images every three minutes for 15 to 24 hours. Download the data and proceed with image analysis in a program of choice such as Matlab.Initially, phase-contrast imaging of cell dots revealed that invasive, lung colony-forming breast cancer cells maintain a mesenchymal phenotype after stimulation with EGF. However, single cells leaving the sheet are observed more frequently in EGF-stimulated cell dots. The response of lung colony-forming breast cancer cells to EGF was further assessed by H and E staining of cultures that were stimulated with EGF for four days.Indeed, EGF stimulation resulted in an increased colony diameter, indicating that migration of EGF-stimulated cells might be altered. In this dot assay, cells at the sheet edges were imaged for nine hours following stimulation of colony-forming breast cancer cells with EGF. PIV revealed that EGF increased cell speed to 0.63 micrometers per minute from 0.45 micrometers per minute in control cells.In addition, EGF reduced the variability of cell directionality, or angular spread, from 0.95 in control cultures to 0.79 in EGF-stimulated cultures. In addition, as was observed by H and E staining after four days, EGF increased the radial displacement of the epithelial edge over nine hours, from 257 micrometers to 356 micrometers. Once mastered, the hands-on time for this technique is a couple of hours.Following this procedure, other methods like time lapse imaging, immunostaining, and DNA and RNA analysis can be performed in order to answer additional questions. For example, it can be used to analyze if proteins are differentially expressed in fast or slow migrating cells, or when cells migrate into the surrounding environment. After watching this video, you should have a good understanding of how to set up the dot assay, and use it to assess the migratory phenotype of attached cohesive cell sheets in response to microenvironemntal cues.

Research

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The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

Ein umfassendes Verfahren zur Bewertung der In-vitro- Leistung von der vermeintlichen Hämangioblastom Neovaskularisation mit den Sphäroid sprießen Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Live Cell Imaging-Assays Studie Glioblastom Gehirn Tumor Stammzellen Migration und Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Echtzeit-Erkennung von In Vitro Tumor Zelle Apoptose induziert durch CD8+ T-Zellen, unterdrückende Immunfunktionen Tumor infiltrieren myeloische Zellen zu studieren

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Eine einfache Migration/Invasion Workflow mit einer automatisierten Leben-Zelle-Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Impedanzbasierte Echtzeitmessung der Krebszellmigration und -invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells

Ein Reporter-Assay zur Analyse der intronischen microRNA-Reifung in Säugetierzellen

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves ...

Quantifizierung der Telomerlänge mittels nicht-radioaktiver Chemilumineszenzdetektion

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Autor im Rampenlicht: Optimierung der Leistungsparameter des TAGGG Telomerlängen-Assays  

Optimierung der Leistungsparameter des TAGGG Telomerlängen-Assays

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...