Name: a simple migration/invasion workflow using an automated live-cell imager
Uploaded: 2019-02-02T14:00:00
Description: Watch this Scientific Journal Video about A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager at JoVE.com

We would like to acknowledge our funding support by the Bloomfield Group Foundation through the Hunter Medical Research Institute (HMRI 13-02). X.Z is supported by an APA scholarship through the University of Newcastle and the HCRA Biomarkers Flagship PhD Scholarship.

NOTE: Two cell lines should be handled separately. The following procedures should be applied to one single cell line if not specified.
1. Optimize Cell Density Prior to Wounding
<ol>
	<li>Culture adherent cells in T75 cm2 tissue culture flasks to about 80% confluence in phenol-red free Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 200 mM L-glutamine and 2 &#181;g/mL insulin at 37 &#176;C with 5% CO2 (standa...

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	<li>Graham, C. H., Lala, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+placental+invasion+of+the+uterus+and+their+control.">Mechanisms of placental invasion of the uterus and their control.</a> Biochemistry and Cell Biology. 70, 867-874 (1992).</li><li>Affolter, M., Zeller, R., Caussinus, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+remodelling+through+branching+morphogenesis.">Tissue remodelling through branching morphogenesis.</a> Nature Reviews Molecular Cell Biology. 10, 831-842 (2009).</li><li>Brugues, 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=Forces+driving+epithelial+wound+healing.">Forces driving epithelial wound healing.</a> Nature Physics. 10, 683-690 (2014).</li><li>Weigelt, B., Peterse, J. L., van't Veer, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+metastasis:+markers+and+models.">Breast cancer metastasis: markers and models.</a> Nature Reviews Cancer. 5, 591-602 (2005).</li><li>Kalluri, R., 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=The+basics+of+epithelial-mesenchymal+transition.">The basics of epithelial-mesenchymal transition.</a> Journal of Clinical Investigation. 119, 1420-1428 (2009).</li><li>Scully, O. J., Bay, B. H., Yip, G., Yu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+metastasis.">Breast cancer metastasis.</a> Cancer Genomics Proteomics. 9, 311-320 (2012).</li><li>Kramer, N., 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=In+vitro+cell+migration+and+invasion+assays.">In vitro cell migration and invasion assays.</a> Mutation Research/Reviews in Mutation Research. 752, 10-24 (2013).</li><li>Holliday, D. L., Speirs, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choosing+the+right+cell+line+for+breast+cancer+research.">Choosing the right cell line for breast cancer research.</a> Breast Cancer Research. 13, 215 (2011).</li><li>Clark, A. G., Vignjevic, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modes+of+cancer+cell+invasion+and+the+role+of+the+microenvironment.">Modes of cancer cell invasion and the role of the microenvironment.</a> Current Opinion in Cell Biology. 36, 13-22 (2015).</li></ol>

Migration and invasion are important parameters to assess the mobility of cancer cells. By using the 96-pin scratch tool, it is possible to conduct wound healing assays in 2D and 3D simultaneously. Apart from facilitating automatic scanning, providing a stable cell culture environment with minimum disruption, the scratch assay conducted using the 96-pin scratch tool provides consistent scratch wounds, enabling experiments that are more robust and reproducible. The 96-well plate format gives additional options of either i...

Cell migration and invasion are important biological processes that enable normal functions in the human body, such as wound closure, invasion of placenta into the uterus and mammary gland morphogenesis1,2,3. The human body has precise and strict control of these biological events; however, there are some exceptions. Malignant tumors, for example, are able to escape this safeguard, exhibit abnormal proliferation and invade into neighboring tissue, which is called metastasis. Metastasis is the major cause of cancer-related mortality4.
Breast cancer is the most commonly diagnosed cancer in women, and is the second-highest cause of cancer-related death among women in developed countries worldwide5. Breast cancer originates from ducts or lobules that consist of one or more layers of epithelial cells. In the normal breast, epithelial cells adhere to one another and to the basement membrane through membrane proteins such as E-cadherin and integrins6. However, invasive breast cancer cells have lost their polarity and cell-cell adhesion, and classically undergo epithelial mesenchymal transition (EMT) and gain the ability to move. After extravasation, these cells move across the extracellular matrix (ECM) and enter the blood vessel or the lymphatic system, followed then by intravasation and metastatic growth7. Understanding the mechanisms by which this occurs is of great significance, since metastasis is the most common cause of cancer-related mortalities and is closely related to cancer cell migration/invasion. To visualize the movement of cancer cells, migration and invasion assays are ideal models to study 2D and 3D cell movement, respectively. Migration directly assesses the movement of the cells whereas invasion involves interaction with the microenvironment and the ability to degrade biological barriers. The two processes are not fully independent of one another, as migration is a requirement of invasion.
Several methods have been developed to study migration and invasion. As reviewed by Kramer et al., migration assays such as wound healing, fence and micro-carrier assays generate a cell-free area to allow cells to move into, assessing the change of area; whereas, transwell and capillary assays are based on the number of cells that move toward an attractant8. For invasion assays, an ECM environment has to be set up with ECM gel or collagen for instance, and 3D movement can be assessed by monitoring the invasion area, distance and cell counts (e.g. transwell assay, platypus assay)8. Another type of invasion assay is to combine the invasive cells with non-invasive cells and assess the behavior of the invasive cells (e.g. spheroid assays). The above methods have their pros and cons, and a way that is easy to approach, easy to repeat, and to combine the migration assay and invasion assay in a similar workflow is preferential in experimental design.
This protocol describes the measurement of cell migration and invasion using a live-cell imager. It is a real-time cell monitoring system installed in a standard cell culture incubator. It takes high definition images according to the set scanning intervals and measurements by applying appropriate masks to the cells or fluorescent targets. The module of migration/invasion assay includes using a 96-pin scratch tool, which is suitable for making homogeneous scratch wounds on a cell monolayer in a 96-well plate. The mechanism is based on in vitro wound healing assays, monitoring 2D cell movement on a plastic or coated surface. Invasion or 3D movement across an additional ECM within the scratch wound can also be assessed. A brief workflow is illustrated in Figure 1.

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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">0.5% trypsin-EDTA solution (10x)</td>
			<td style="width:100px;">ThermoFisher Scientific</td>
			<td style="width:119px;">30028-02</td>
			<td style="width:167px;">Dilute to 2x in DPBS</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Countess II FL Automated Cell Counter</td>
			<td style="width:100px;">ThermoFisher Scientific</td>
			<td style="width:119px;">AMQAX1000</td>
			<td style="width:167px;">Automated cell counter</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Detergent 1 (Alconox)</td>
			<td style="width:100px;">Sigma-Aldrich</td>
			<td style="width:119px;">242985</td>
			<td style="width:167px;">0.5% working concentration</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Detergent 2 (Virkon S)</td>
			<td style="width:100px;">VetProduct DIRECT</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">1% working concentration</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:309px;">Dulbecco&#8217;s Modified Eagle Medium, no phenol-red</td>
			<td style="width:100px;">ThermoFisher Scientific</td>
			<td style="width:119px;">21063-045</td>
			<td style="width:167px;">Supplimented with 10% FBS, 200 mM L-glutamine, 2 &#956;g/ml insulin</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">ECM Gel (matrigel)</td>
			<td style="width:100px;">Sigma-Aldrich</td>
			<td style="width:119px;">E6909</td>
			<td style="width:167px;">Growth-factor reduced, phenol red free</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Essen ImageLock 96-well plate, flat bottom</td>
			<td style="width:100px;">Essen</td>
			<td style="width:119px;">4379</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">EVE Counting slides</td>
			<td style="width:100px;">BioTools</td>
			<td style="width:119px;">EVS-50</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Fetal bovine serum (FBS)</td>
			<td style="width:100px;">Bovogen Biologicals</td>
			<td style="width:119px;">SFBS-F-500ml</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">IncuCyte 96-well scratch wound cell invasion accessories</td>
			<td style="width:100px;">Essen</td>
			<td style="width:119px;">4444</td>
			<td style="width:167px;">Including CoolBox, 2x CoolSink</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:309px;">IncuCyte Cell migration kit</td>
			<td style="width:100px;">Essen</td>
			<td style="width:119px;">4493</td>
			<td style="width:167px;">Including the 96-well pin block, 2x wash boats and the software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">IncuCyte ZOOM</td>
			<td style="width:100px;">Essen</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Live cell analysis system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Insulin solution human</td>
			<td style="width:100px;">Sigma-Aldrich</td>
			<td style="width:119px;">19278-5ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">L-glutamine solution (100x)</td>
			<td style="width:100px;">ThermoFisher Scientific</td>
			<td style="width:119px;">25030-091</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Tissue culture flask, 75 cm2 growth area</td>
			<td style="width:100px;">Greiner Bio-One</td>
			<td style="width:119px;">658175</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

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.

This migration/invasion assay is based on the wound healing assay, which evaluates the rate of the cells moving into a cell-free area created by the 96-pin scratch tool. The difference between the migration and invasion assays are that migration assays measure cells moving on the tissue-culture treated plastic surface and invasion measures cells moving across ECM gel.
The scratch tool is designed to make consistent scratch wound...

Watch this Scientific Journal Video about A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager at JoVE.com

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

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 ...

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