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

O autor deseja agradecer lucianoteixeira Subramanian, Chang Yi (Jason), Paul Randazzo e Carole Parent para ler e comentar sobre o manuscrito. Este trabalho foi financiado pelo programa de pesquisa Intramural do Instituto Nacional de câncer, National Institutes of Health.

1. revestimento de pratos (dia 1)
Nota: Certifique-se para não deixar impressões digitais ou sujeira na parte inferior da placa, quando manuseá-lo.
<ol><li>Descongelar o colágeno rato IV no gelo e dilui-lo com 50mm HCl (pH 1,3) para preparar 3 mL de solução 10 µ g/mL colagénio IV. Nota: Colágeno irá precipitar a 37 ° C. Portanto, é importante manter a solução de colágeno em uma baixa temperatura ao descongelamento e trabalhar com ele. Evite ciclos repetidos de congelamento-descongelamento por p...

<ol>
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H., Busch, J. I., 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=Transient+tumor-fibroblast+interactions+increase+tumor+cell+malignancy+by+a+TGF-Beta+mediated+mechanism+in+a+mouse+xenograft+model+of+breast+cancer.">Transient tumor-fibroblast interactions increase tumor cell malignancy by a TGF-Beta mediated mechanism in a mouse xenograft model of breast cancer.</a> PLOS ONE. 5 (3), 9832 (2010).</li><li>Normanno, N., De Luca, A., 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=Epidermal+growth+factor+receptor+(EGFR)+signaling+in+cancer.">Epidermal growth factor receptor (EGFR) signaling in cancer.</a> Gene. 366 (1), 2-16 (2006).</li><li>Harrison, S. M. W., Knifley, T., Chen, M., O'Connor, K. 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. 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Durante as células de progressão do tumor migre longe do tecido de origem para invadir o tecido circundante e metástase para locais distantes15. Migração de células de uma colônia de células pode ser observada no ensaio de ponto. Aqui, o ensaio de ponto é ilustrado por analisar o fenótipo migratório de células de câncer de mama humano em resposta a EGF.
Para obter resultados precisos e replicáveis várias etapas na configuração do ponto os ensaios são c...

Ensaios de migração são amplamente utilizados para avaliar o potencial invasivo e metastático do tumor células em vitro. Mais comumente, a ferida ou ensaio zero é usado para avaliar a migração de folhas epiteliais em uma célula desmarcada área1,2,3. Para realizar o ensaio de zero, as células são banhadas em um monolayer e uma área livre de célula ou "zero" é criada com uma ponta da pipeta. O ensaio de zero é fácil de configurar com suprimentos de cultura de tecidos comumente disponíveis e pode ser executado em placas multi bem, permitindo processamento de amostras múltiplas. No entanto, como é feito o zero, células são fisicamente removidas a monocamada e muitas vezes passam por morte celular. Além disso, ligada à placa de matriz extracelular é muitas vezes danificada durante o processo de coçar. Da mesma forma, o uso de silício insere (tais como câmaras de Ibidi4) ou de estênceis5,6,7 pode levar a ruptura mecânica das células e a remoção parcial de proteínas da matriz usada para revestimento a placas. Outra desvantagem dos ensaios de acompanhamento de fechamento de ferimentos ou arranhões é seu curso de tempo limitado, como migração celular só pode ser analisada até que o zero é fechado.
Realizar o ensaio de ponto, as células são banhadas como uma colônia circular sobre uma placa revestidos ou não revestidos de8. A justificativa para esta estratégia de chapeamento é obter lençóis de células com bordas definidas, que podem migrar ou invadir em áreas circundantes sem célula sem perturbar a cultura pela remoção de células ou inserções. O objetivo geral do ensaio ponto é observar a migração das folhas de célula medida pelo deslocamento de borda ou diâmetro colônia, bem como para realizar a imagem de lapso de tempo para analisar o fenótipo migratório das células em maior resolução espaço-temporal.
Migração celular pode ser afetada por uma variedade de sugestões microenvironmental como fatores de crescimento como EGF, citocinas e quimiocinas. EGF é um fator de crescimento que exerce seus efeitos biológicos através da ligação ao seu receptor, o receptor de EGF9e aumenta o comportamento invasivo e metastático de células de tumor a4,9,10. Aqui, o ensaio de ponto é usado para estudar EGF estimulou a migração de células em uma colônia de pulmão humano invasoras, formando mama câncer célula linha (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

Apesar de organismos complexos aparecem estáticos, seus tecidos estão sob contínua um volume de negócios. Como células de idade morrem e são substituídas por novas células, as células mover dentro de tecidos de forma bem orquestrada. Durante o desenvolvimento do tumor, este equilíbrio é perturbado, e células tumorais deixar o epitélio de origem para invadir o local microambiente, para viajar para locais distantes e, finalmente, formar tumores metastáticos em locais distantes. O ensaio de ponto é um ensaio de migração irrestrita simples, bidimensional, para avaliar o movimento líquido de folhas de célula em uma área livre de célula e analisar parâmetros de migração celular utilizando imagens de lapso de tempo. Aqui, o ensaio de ponto é demonstrado usando um humano invasivo, formadoras de pulmão em linhagem de células de câncer de mama, MCF10CA1a, para analisar a resposta de migratória das células para fator de crescimento epidérmico (EGF), que é conhecida por aumentar o potencial maligno das células de câncer de mama e para alterar o fenótipo migratório das células.

O ensaio de ponto aqui apresentado (Figura 1) foi realizado utilizando invasiva, colônia de pulmão formando células de câncer de mama (MCF10CA1a) como um sistema modelo. O ensaio de ponto rende pontos de célula altamente reprodutível nem na mão de iniciantes, tornando-se um conveniente e fácil de executar o ensaio (Figura 1D). O ensaio de ponto combinado com ele mancha, ou lapso de tempo de imagem e subse...

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

Usando o ensaio de ponto para analisar a migração de células de folhas

Migração celular é essencial para o desenvolvimento, manutenção de tecidos e reparação e tumorigênese e é regulada por citocinas, quimiocinas e fatores de crescimento. Este protocolo descreve o ensaio de ponto, um ensaio de migração bidimensional, irrestrita para avaliar o fenótipo migratório de folhas de célula unida, coesa, em resposta a sinais microenvironmental.

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.

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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 ...

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

Um procedimento abrangente para avaliar o desempenho In Vitro da neovascularização de Hemangioblastoma putativo usando o Spheroid brotando do ensaio

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

Imagem latente da viver-pilha ensaios de migração de células-tronco do estudo Glioblastoma cerebral Tumor e invasão

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

Detecção de tempo real em Vitro Tumor celular apoptose induzida por CD8+ T células para estudar as funções imunológicas supressora de Tumor infiltrando células mieloides

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

Um fluxo de trabalho simples migração/invasão usando um automatizado Imager viver-pilha

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

Medição em tempo real da migração e invasão de células cancerígenas

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

Um ensaio repórter para analisar a maturação intronic microRNA em células mamíferas

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

Quantificando o comprimento dos telômeros usando detecção de quimioluminescência não radioativa

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Autor em destaque: Otimização dos parâmetros de desempenho do ensaio de comprimento dos telômeros TAGGG  

Otimização dos parâmetros de desempenho do ensaio de comprimento telomérico TAGGG

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