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 them at -80 &#176;C.</li>
	<li>Add 250 &#181;L collagen IV solution to each well 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. Incubate the plates overnight at room temperature. 
	Note: As only the growth area needs to be coated, it is sufficient to only cover the cover slip, which is attached to the bottom of the plate, with collagen solution (see Figure 1A, B for a schematic of plate).</li>
	<li>Next morning, rinse plates with deionized H2O twice to remove non-absorbed collagen and buffer. Direct the water to the edge of the plate well, not to the edge formed by the plate bottom and the coverslip (if water is pipetted into this area it will splash). Air-dry the plates in the laminar flow hood. Use plates immediately or store at 4 &#176;C for up to 5 days. 
	Note: Different cell lines might require different coating, such as fibronectin or collagen I.</li>
</ol>
2. Plating the Dot Assay (Day 2)
<ol>
	<li>Preparing the cell suspension
	<ol>
		<li>To prepare cell suspension, use cells that have been grown in a 60-mm tissue culture dish in DMEM/F12 medium supplemented with 5% horse serum to 80% confluence</li>
		<li>Rinse the cells with calcium- and magnesium-free Dulbecco&#39;s phosphate buffered saline (DPBS) once, add 500 &#181;L trypsin-EDTA (room temperature), and incubate the cells for 3 - 5 min in an incubator at 37 &#176;C, 5% CO2, humidified atmosphere.</li>
		<li>Suspend the detached cells in 4.5 mL culture medium to stop trypsin activity and count a 20 &#181;L aliquot of the cell suspension in a hemocytometer.</li>
		<li>Pellet the remaining cells in a 15 mL conical tube by centrifugation at 200 x g for 3 min, aspirate the supernatant, and resuspend the cells in culture medium to 3 x 106 cells/mL. 
		NOTE: For other coating substrates and/or cell lines, the optimal cell density must be established in a dilution series. 3 x 106 cell/mL is recommended as a starting point.</li>
	</ol>
	</li>
	<li>Plating cells
	<ol>
		<li>Place a 10 &#181;L drop of cell suspension at the center of each cover slip of the collagen IV coated 12-well glass bottom plate without touching or scratching the coating (Figure 1A, B). Incubate the plate for 30 min at 37 &#176;C, humidified atmosphere, to allow the cells to attach. Check in an inverted microscope that the cells are attached. 
		NOTE: This can be best seen at the edge of the drop, where formation of a monolayer can be observed (Figure 1C). If necessary, incubate the plate up to 3 h. Be sure that the humidity in the incubator is high enough as the drops can dry out quickly. If a reduction of the drop volume during this step is observed, add PBS to the spaces between the wells to increase humidity.</li>
		<li>Gently wash the wells with 1 mL of culture medium twice to remove non-attached cells. Add 1 mL culture medium to each well. Check in an inverted microscope that no floating cells remain, otherwise wash the wells again. Incubate cells overnight at 37 &#176;C, 5% CO2, humidified atmosphere, to obtain well-defined cell sheets. 
		Note: Floating cells are likely to re-attach to the plate and to form undesired cell colonies.</li>
	</ol>
	</li>
</ol>
3. Stimulating Cells (Day 3)
<ol>
	<li>Check all wells using an inverse microscope to ensure that cell colonies have grown properly. If necessary, mark unsuitable wells and do not use them.</li>
	<li>Label each well of the plate appropriately for each condition investigated. Run each condition in duplicate or triplicate.</li>
	<li>Change the medium to starve the cells in DMEM/F12 containing 0.1% horse serum (starving medium) 3 h before cells are stimulated.</li>
	<li>Stimulate the cells by addition of EGF (5 ng/mL final concentration) or other desired mediators and gently mix the medium using a 1 mL micropipettor or by swirling the plate.</li>
	<li>Incubate the plate for 1 - 4 days to observe edge displacement and edge contour as described in section 4.1 or perform time-lapse imaging as described in section 4.2.</li>
</ol>
4. Visualization of Cell Colonies and Cell Migration
<ol>
	<li>Hematoxylin-eosin (HE) staining to measure colony growth (Day 4 - 7)
	<ol>
		<li>Fix cells in 70% ethanol (1 mL/well) for ~2 min.</li>
		<li>Stain nuclei with hematoxylin (1 mL/well), ~2 min.</li>
		<li>Wash cells with tap water (1 mL/well) until nuclei are blue, 5 - 10 min.</li>
		<li>Stain cytoplasm with eosin (1 mL/well), ~2 min.</li>
		<li>Rinse with deionized water (1 mL/well). 
		Note: Hematoxylin and eosin solutions can be collected and used multiple times until the staining becomes faint.</li>
		<li>Air-dry plate.</li>
		<li>Flip the plate and measure the dot diameter with a ruler. Alternatively, take pictures of the plate with a camera, and analyze the dot diameter in ImageJ.</li>
	</ol>
	</li>
	<li>Time-lapse microscopy (Day 3)
	<ol>
		<li>Switch on the incubator microscope and adjust the temperature to 37 &#176;C. Fill the humidifier with dH2O. Adjust the CO2 to 5%. Ensure that the incubator chamber is humidified and that the motorized stage can move freely. Close the incubator chamber and allow the microscope to adjust to 37 &#176;C. 
		NOTE: The microscope (see the Table of Materials) used here should be heated to 37 &#176;C for at least 3 h before the cells are imaged to avoid drifting of the focus.</li>
		<li>Place the plate on the stage of the incubator microscope and set up the stage list. Image two opposing edges and the center of each cell dot (Figure 1A).</li>
		<li>Take images every 3 min, for 15 - 24 h using the 10X&#160;objective. Download the data and proceed with image analysis in a program of choice, e.g., ImageJ or Matlab.</li>
	</ol>
	</li>
</ol>

<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. L. <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+and+invasion+assays+as+tools+for+drug+discovery.">Cell migration and invasion assays as tools for drug discovery.</a> Pharmaceutics. 3 (1), 107-124 (2011).</li><li>Weiger, M. C., Vedham, V., 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=Real-time+motion+analysis+reveals+cell+directionality+as+an+indicator+of+breast+cancer+progression.">Real-time motion analysis reveals cell directionality as an indicator of breast cancer progression.</a> PLOS ONE. 8 (3), 58859 (2013).</li><li>Poujade, M., Grasland-Mongrain, E., 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=Collective+migration+of+an+epithelial+monolayer+in+response+to+a+model+wound.">Collective migration of an epithelial monolayer in response to a model wound.</a> Proc Natl Acad Sci U S A. 104 (41), 15988-15993 (2007).</li><li>Sunyer, R., Conte, V., 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=Collective+cell+durotaxis+emerges+from+long-range+intercellular+force+transmission.">Collective cell durotaxis emerges from long-range intercellular force transmission.</a> Science. 353 (6304), 1157-1161 (2016).</li><li>Bazelli&#232;res, E., Conte, V., 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=Control+of+cell-cell+forces+and+collective+cell+dynamics+by+the+intercellular+adhesome.">Control of cell-cell forces and collective cell dynamics by the intercellular adhesome.</a> Nat Cell Biol. 17 (4), 409-420 (2015).</li><li>Stuelten, C. 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. 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The author has nothing to disclose.

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 ...

using-the-dot-assay-to-analyze-migration-of-cell-sheets

Research

JoVE Journal

Cancer Research

6.8K Views.  National Institutes of Health. 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.

Video: Using the Dot Assay to Analyze Migration of Cell Sheets

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention regimens.

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the beginning and end of an experiment using a phase-contrast microscope, and the migration abilities of cells are evaluated by the closure of wounds. However, cell movement is a dynamic phenomenon, and a conventional method does not allow for tracking single-cell movement. To improve current wound-healing assays, we use live-cell imaging techniques to monitor cell migration in real time. This method allows us to determine the cell migration rate based on a cell tracking system and provides a clearer distinction between cell migration and cell proliferation. Here, we demonstrate the use of live-cell imaging in wound-healing assays to study the different migration abilities of breast epithelial cells influenced by the presence of TIP60. As cell motility is highly dynamic, our method provides more insights into the processes of wound healing than a snapshot of wound closure taken with the traditional imaging techniques used for wound-healing assays.

Here, we present the real-time monitoring of cell migration in a wound-healing assay using TIP60-depleted MCF10A breast epithelial cells. The implementation of live-cell imaging techniques in our protocol allows us to analyze and visualize single-cell movement in real time and across time.

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the ...

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an &#34;in-incubator&#34; video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.

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

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human central nervous system (CNS). However, both the cytological origin and the evolutionary process of HBs (including neovascularization) remain controversial, and anti-angiogenesis for VHL-HBs, based on classic HB angiogenesis, have produced disappointing results in clinical trials. One major obstacle to the successful clinical translation of anti-vascular treatment is the lack of a thorough understanding of neovascularization in this vascular tumor. In this article, we present a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in HBs, as well as its role in HBs. With this procedure, researchers can accurately understand the complexity of HB neovascularization and identify the function of this common form of angiogenesis in HBs. These protocols can be used to evaluate the most promising anti-vascular therapy for tumors, which has high translational potential either for tumors treatment or for aiding in the optimization of the anti-angiogenic treatment for HBs in future translations. The results highlight the complexity of HB neovascularization and suggest that this common form angiogenesis is only a complementary mechanism in HB neovascularization.

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

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

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

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

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

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

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

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

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

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

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

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental disorders. A characterization of disease-related RAF mutants will help us select appropriate therapeutic strategies for treating these diseases. Recent studies have shown that RAF family kinases have both catalytic and allosteric activities, which are tightly regulated by dimerization. Here, we constructed a set of practical and feasible methods to determine the catalytic and allosteric activities and the relative dimer affinity/stability of RAF family kinases and their mutants. Firstly, we amended the classical in vitro kinase assay by reducing the detergent concentration in buffers, utilizing a gentle quick wash procedure, and employing a glutathione S-transferase (GST) fusion to prevent RAF dimers from dissociating during purification. This enables us to measure the catalytic activity of constitutively active RAF mutants appropriately. Secondly, we developed a novel RAF co-activation assay to evaluate the allosteric activity of kinase-dead RAF mutants by using N-terminal truncated RAF proteins, eliminating the requirement of active Ras in current protocols and thereby achieving a higher sensitivity. Lastly, we generated a unique complementary split luciferase assay to quantitatively measure the relative dimer affinity/stability of various RAF mutants, which is more reliable and sensitive compared to the traditional co-immunoprecipitation assay. In summary, these methods have the following advantages: (1) user-friendly; (2) able to carry out effectively without advanced equipment; (3) cost-effective; (4) highly sensitive and reproducible.

In this article, we presented a set of practical and feasible methods for characterizing disease-related mutants of RAF family kinases, which include in vitro kinase assay, RAF co-activation assay, and complementary split luciferase assay.

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

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

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These acquired abnormalities in complex, multilayered molecular signaling networks induce aberrant cell proliferation and survival, extracellular matrix degradation, and metastasis to distant organs. Approximately 90% of cancer-related deaths are estimated to be caused by the direct or indirect effects of metastatic dissemination. Therefore, it is important to establish a highly reliable, comprehensive system to characterize cancer cell behaviors upon genetic and environmental manipulations. Such a system can give a clear understanding of the molecular regulation of cancer metastasis and the opportunity for successful development of stratified, precise therapeutic strategies. Hence, accurate determination of cancer cell behaviors such as migration and invasion with gain or loss of function of gene(s) allows assessment of the aggressive nature of cancer cells. The real-time measurement system based on cell impedance enables researchers to continually acquire data during a whole experiment and instantly compare and quantify the results under various experimental conditions. Unlike conventional methods, this method does not require fixation, staining, and sample processing to analyze cells that migrate or invade. This method paper emphasizes detailed procedures for real-time determination of migration and invasion of glioblastoma cancer cells.

Cancer is a lethal disease due to its ability to metastasize to different organs. Determining the ability of cancer cells to migrate and invade under various treatment conditions is crucial to assessing therapeutic strategies. This protocol presents a method to assess the real-time metastatic abilities of a glioblastoma cancer cell line.&#160;

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

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves different RNA-binding proteins in the nucleus. Mature miRNAs frequently mediate gene silencing, and this has become an important tool for comprehending post-transcriptional events. Besides that, it can be explored as a promising methodology for gene therapies. However, there is currently a lack of direct methods for assessing miRNA expression in mammalian cell cultures. Here, we describe an efficient and simple method that aids in determining miRNA biogenesis and maturation through confirmation of its interaction with target sequences. Also, this system allows the separation of exogenous miRNA maturation from its endogenous activity using a doxycycline-inducible promoter capable of controlling primary miRNA (pri-miRNA) transcription with high efficiency and low cost. This tool also allows modulation with RNA-binding proteins in a separate plasmid. In addition to its use with a variety of different miRNAs and their respective targets, it can be adapted to different cell lines, provided these are amenable to transfection.

We developed an intronic microRNA biogenesis reporter assay to be used in cells in vitro with four plasmids: one with intronic miRNA, one with the target, one to overexpress a regulatory protein, and one for Renilla luciferase. The miRNA was processed and could control luciferase expression by binding to the target sequence.

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