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

The author wishes to thank Bhagawat Subramanian, Yi (Jason) Chang, Paul Randazzo, and Carole Parent for reading and commenting on the manuscript. This work was supported by the Intramural Research Program of the National Cancer Institute, National Institutes of Health.

1. Coating of Dishes (Day 1)
NOTE: Make sure not to leave fingerprints or dirt on the bottom of the plate when handling it.
<ol>
	<li>Thaw mouse collagen IV on ice, and dilute it with 50 mM HCl (pH 1.3) to prepare 3 mL of a 10 &#181;g/mL collagen IV solution. 
	NOTE: Collagen will precipitate at 37 &#176;C. It is therefore important to keep the collagen solution at a low temperature while thawing and working with it. Avoid repeated freeze-thaw cycles by preparing appropriately sized aliquots and storing the...

<ol>
	<li>Liang, C. -. C., Park, A. Y., Guan, J. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+scratch+assay:+a+convenient+and+inexpensive+method+for+analysis+of+cell+migration+in+vitro.">In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro.</a> Nat Protoc. 2 (2), 329-333 (2007).</li><li>Peplow, P. V., Chatterjee, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+influence+of+growth+factors+and+cytokines+in+in+vitro+human+keratinocyte+migration.">A review of the influence of growth factors and cytokines in in vitro human keratinocyte migration.</a> Cytokine. 62 (1), 1-21 (2013).</li><li>Hulkower, K. I., Herber, R. 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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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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+density+determines+epithelial+migration+in+culture.">Cell density determines epithelial migration in culture.</a> Proc Natl Acad Sci U S A. 77 (8), 4760-4763 (1980).</li></ol>

During tumor progression cells migrate away from the tissue of origin to invade the surrounding tissue and metastasize to distant sites15. Migration of cells away from a cell colony can be observed in the dot assay. Here, the dot assay is illustrated by analyzing the migratory phenotype of human breast cancer cells in response to EGF.
To obtain accurate and replicable results several steps in the set-up of the dot assays are critical. First, the coating of the plate det...

Migration assays are widely used to evaluate the invasive and metastatic potential of tumor cells in vitro. Most commonly, the wound or scratch assay is used to assess migration of epithelial sheets into a cell cleared area1,2,3. To perform the scratch assay, cells are plated into a monolayer and a &#34;scratch&#34; or cell-free area is created with a pipette tip. The scratch assay is easy to set up with commonly available tissue culture supplies and can be performed in multi-well plates, allowing for processing of multiple samples. However, as the scratch is made, cells are physically removed from the monolayer and often undergo cell death. Furthermore, extracellular matrix attached to the plate is often damaged during the scratching process. Similarly, the use of silicon inserts (such as Ibidi chambers4) or of stencils5,6,7 can lead to mechanical disruption of the cells and the partial removal of matrix proteins used for coating the plates. Another disadvantage of assays monitoring closure of wounds or scratches is their limited time course, as cell migration can only be analyzed until the scratch is closed.
In performing the dot assay, cells are plated as a circular colony onto a coated or uncoated plate8. The rationale for this plating strategy is to obtain cell sheets with defined edges, that can migrate or invade into the surrounding cell-free areas without disturbing the culture by removal of cells or inserts. The overall goal of the dot assay is to observe migration of cell sheets as measured by edge displacement or colony diameter, as well as to perform time-lapse imaging to analyze the migratory phenotype of cells in higher spatio-temporal resolution.
Cell migration can be affected by a variety of microenvironmental cues like chemokines, cytokines, and growth factors such as EGF.&#160;EGF is a growth factor that exerts its biological effects via binding to its receptor, EGF receptor9, and increases invasive and metastatic behavior of tumor cells4,9,10. Here, the dot assay is used to study EGF stimulated cell migration in a human invasive, lung colony forming breast cancer cell line (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

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move within tissues in a tightly orchestrated manner. During tumor development, this equilibrium is disturbed, and tumor cells leave the epithelium of origin to invade the local microenvironment, to travel to distant sites, and to ultimately form metastatic tumors at distant sites. The dot assay is a simple, two-dimensional unconstrained migration assay, to assess the net movement of cell sheets into a cell-free area, and to analyze parameters of cell migration using time-lapse imaging. Here, the dot assay is demonstrated using a human invasive, lung colony forming breast cancer cell line, MCF10CA1a, to analyze the cells' migratory response to epidermal growth factor (EGF), which is known to increase malignant potential of breast cancer cells and to alter the migratory phenotype of cells.

The dot assay presented here (Figure 1) was performed using invasive, lung colony forming breast cancer cells (MCF10CA1a) as a model system. The dot assay yields highly reproducible cell dots even in the hand of beginners, making it a convenient and easy to execute assay (Figure 1D). The dot assay combined with HE staining, or time-lapse imaging and subsequent particle image velocimetry (PIV) allows the&#160;stud...

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

Cell migration is essential for development, tissue maintenance and repair, and tumorigenesis, and is regulated by growth factors, chemokines, and cytokines. This protocol describes the dot assay, a two-dimensional, unconstrained migration assay to assess the migratory phenotype of attached, cohesive cell sheets in response to microenvironmental cues.

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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