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 (standard incubation conditions for most cancer cell lines, culture formulations are cell line-dependent).</li>
	<li>Remove the culture media by pipetting off into a waste container. Pipet 2 mL of pre-warmed 0.1% trypsin-EDTA to briefly rinse the cell monolayer and pipet off. Add another 2 mL of 0.1% trypsin-EDTA and place the flask under standard incubation conditions at 37 &#176;C for 5 min.</li>
	<li>Gently tap the flask to ensure detachment of the cells and then add 10 mL of pre-warmed culture media to stop the proteolytic reaction.</li>
	<li>Transfer the cell suspension into a 15 mL centrifuge tube and spin down at 200 x g for 5 min at room temperature (RT). Carefully remove the supernatant without agitating the cell pellet and resuspend with another 10 mL pre-warmed culture media.</li>
</ol>
2. Counting Cell Number Using an Automated Cell Counter (or Any Counting Methods)
<ol>
	<li>Dilute 200 &#181;L of resuspended cell suspension with 800 &#181;L of 1x Dulbecco&#39;s Phosphate buffered Saline (DPBS) in a 1.5 mL centrifuge tube.</li>
	<li>Attach a 60 &#181;m cell count sensor to the cell counter, hold down the toggle and merge the tip into the cell suspension. Slowly release the toggle until the cell suspension is successfully drawn into the sensor. Cell concentration is displayed in cells/mL.</li>
	<li>Calculate the cell number in the 10 mL cell suspension.</li>
</ol>
3. Cell Plating
<ol>
	<li>Plate cells at a range of cell densities (40,000-90,000 cells/well) in triplicate in a 96-well plate.</li>
	<li>Place the plate into the live-cell imager, and schedule scanning every 2 h for 24 h.
	<ol>
		<li>In the software, click Schedule scans from the task list. In the drawer setup pane, determine the position of the plate, click Add vessel and choose the plate type. In the Scan Setup pane, choose or edit the scan pattern according to the experimental plate setup, and set Scan Type as Standard.</li>
		<li>Right click on the Timeline and select Set Intervals. Set Add Scans Every to 2 h at a 24-h schedule. Click Apply.</li>
	</ol>
	</li>
</ol>
4. Determine the Optimal Cell Seeding Density for the Migration Assay
<ol>
	<li>Stop scanning after 24 h, apply the confluence processing analysis tool to the HD-phase contrast images automatically collected, and generate a cell proliferation curve against time.
	<ol>
		<li>Determine 3-6 representative images and place them in a new Image Collection.</li>
		<li>Determine a proper mask as Processing Definition.</li>
		<li>Launch an analysis job.</li>
		<li>Determine optimized cell density according to confluence against time (approximately 100% confluence within 6 -18 h depending on when the migration assay commences). 
		NOTE: The amount of time it takes to grow the cells to confluence varies depending on the seeding dilution.</li>
	</ol>
	</li>
</ol>
5. Days 1 and 2: Preparation for Migration and Invasion Assays
<ol>
	<li>On Day 1, coat the plate for invasion assay. 
	NOTE: ECM gel should always be handled on ice and with tips that have been placed in the fridge overnight.
	<ol>
		<li>Dilute ECM gel with ice-cold culture media to 100 &#181;g/mL and add 50 &#181;L of diluted ECM gel/media into designated wells.</li>
		<li>Place the plate at standard incubation conditions overnight.</li>
	</ol>
	</li>
	<li>On day 2, gently aspirate the excess media. Plate cells with optimized cell densities into 2 96-well plates designated for the migration assay (uncoated) and the invasion assay (coated) in triplicates following sections 1-3 in the late afternoon (in the current protocol, optimized seeding densities for ZR75-1 and MDA-MB-231 were 90,000/well and 50,000/well, respectively).</li>
	<li>Place plates at standard incubation conditions overnight.</li>
</ol>
6. Day 3: Wound Scratch
<ol>
	<li>Spray and wipe the scratch tool and 2 washing boats with 70% ethanol before placing them in the biosafety cabinet. Fill the washing boat 1 and 2 with exactly 45 mL of sterile (autoclaved) distilled water and 70% ethanol respectively.</li>
	<li>For sterilization, place the scratch tool pin block (top) on washing boat 1 and 2 for 5 min each.</li>
	<li>Start with the migration assay plate.
	<ol>
		<li>Move the plate containing cells from the incubator and make sure no well is dry to avoid damage of the scratch tool. Remove the plate cover and insert into the base plate holder of the scratch tool, and carefully place the top part onto the base part by guiding dowels. Press and hold the black lever, and meanwhile carefully lift the pin block. Gaps in each well are usually visible with the naked eye and under the microscope.</li>
		<li>Quickly soak the pins in water; this is sufficient to clean the scratch tool prior to scratching the invasion assay plate if plate setup is identical. Otherwise, repeat the sterilization steps with sterile distilled water and then 70% ethanol for 5 min each.</li>
	</ol>
	</li>
	<li>Wash the plate 1 or 2 times with pre-warmed culture media to avoid detached cells or cell sheets reattaching to the well.</li>
	<li>Add 100 &#181;L of fresh warm media into designated wells with or without treatments.</li>
	<li>Additional steps for invasion assay.
	<ol>
		<li>Place the invasion assay plate at 4 &#176;C for 5 min to equilibrate and carefully aspirate the cold media.</li>
		<li>Dilute ECM gel with ice-cold culture media to 5 mg/mL, add 50 &#181;L of diluted ECM gel into designated wells, and place under standard incubation for 30 min.</li>
		<li>Add 100 &#181;L of warmed-up media with or without testing compounds.</li>
	</ol>
	</li>
	<li>Place the plate into live-cell imager and let it equilibrate for 5 min. Choose Vessel Type as imagelock plate. Set Scan Type as Scratch Wound, choose or edit Scan Pattern according to the experimental plate setup (1 image/well and wide mode) and schedule 24-h repeat scanning every 1-2 h for 72 h until the wounds are healed.</li>
	<li>To clean the scratch tool, put the top pin block in each of the following solutions (45 mL in washing boats) for 5 min: 0.5% detergent 1 (see Table of Materials), 1% detergent 2 (see Table of Materials), sterile distilled water and 70% ethanol. Place the scratch tool back onto its base plate and store in a dust-free environment.</li>
</ol>
7. Data Analysis
<ol>
	<li>Stop scanning the designated plate after all the wounds have healed by choosing the experimental plate on the Drawer Setup and clicking Remove Vessel.</li>
	<li>Collect 3-6 representative images spanning a range of sound percentages, including images right after the wound has been made, wound closure by 10% and 50%.</li>
	<li>To determine a proper processing definition, use Segmentation Adjustment, Cleanup and Filters to apply appropriate Scratch Wound Mask and Confluence Mask. Use Preview current/all to view the accuracy of the masks.</li>
	<li>Launch the analysis job.</li>
	<li>Data can be analyzed within the software or exported for further analysis. Three metrics provided by the software can be used to evaluate the HD-phase images: wound width (&#181;m), wound confluence (%) and relative wound density (%). Comparisons will be discussed in the following section.</li>
</ol>

<ol>
	<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>

The authors declare that they have no competing financial interests.

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 increasing the number of cell lines or different drug treatments. In addition, cell morphology changes and dynamic movement can be recorded for further analysis.
The migration/invasion assay is designed to be easily applicable with a few prerequisites. A confluent cell monolayer is critical prior to wounding to ensure a directed cell movement toward the gap. It is therefore highly recommended to optimize cell seeding density, and a timeframe between 6-18 h&#160;is typically ideal. The ability of the experimental cells attaching to the surface and the proliferation rate will affect this timeframe. Prolonged incubation time may lead to strong cell-cell adhesion and generate crooked bilateral cell fronts due to removal of cell sheets. A biomatrix coating will be helpful with less adhesive cell lines, to decrease the waiting time. Cells can be starved before wounding. Starvation timing is similar to scratch timing, which is when the confluence reaches nearly 100%, and the starvation media and duration are cell type dependent. Cells can also be transfected, but seeding density has to be determined for a prolonged time before wounding, considering incubation time of the transfection process. Wounding can be achieved within a few seconds using the 96-pin scratch tool. Defined-sized pins guarantee a precise migration/invasion starting line and distance. Importantly, the top and bottom sections must be well-aligned to complete a full scratch across the wells, so that the live-cell images don&#39;t include either terminal of the scratch wounds. The scratch wounds can be easily observed through naked eyes on the confluent cell monolayer and can be quickly checked under a bright field microscope. Washing of the wells should be performed immediately and gently to avoid reattachment of dislodged cells and removal of bilateral cells. If one washing is sufficient to clean up the desired cell-free zone, there is no need for an additional wash.
Migration assays can be finalized by adding culture media with or without treatments and are ready to scan, whereas a few additional steps are needed to be taken into account for an invasion assay. Unlike cell migration, invading cells penetrate through ECM, which more closely reflects in vivo cell movement better. The principal of the current protocol is to generate an ECM-surrounded environment by using ECM gel. Cells are seeded on ECM gel-coated wells and after wounding, ECM gel is layered on the cells and the scratch wound. When handling ECM gel, it is better to keep everything cold to avoid gelation. Unevenly distributed ECM gel can cause problems when focusing the microscope. Due to the viscous nature of ECM gel, it is possible to generate bubbles, but the bubbles can be easily removed by aspirating 70% ethanol through a spray bottle. To generate fine but not excessive spray, simply unscrew the cap of the spray bottle and spray off the excess 70% ethanol within the straw pointing elsewhere. By spraying at a distance above and horizontal to the plate, the fine 70% ethanol spray is sufficient to eliminate the bubbles.
The first scan should commence as soon as possible following wounding, to capture the initial cell-free wound area, and this is important for establishing the criteria for the analysis. The scan intervals depend on the cell type, as more invasive cell lines have a more rapid wound closure rate.
This migration/invasion application is relatively straightforward, and by using the built-in analysis module, cell movement can be graphed kinetically with 3 metrics: the wound width, wound confluence and the relative wound density (Figure 3). All three can be used to assess migration. Wound width describes the average distance in &#181;m when the bilateral cell sheets move relatively parallel, and is independent of the initial wound boundary. Wound confluence and relative wound density, on the contrary, require the initial wound boundary in the calculation, and describe the confluence within the wound area and the cell density relative to the unwounded area, respectively. For the invasion assay, relative wound density is recommended, since it computes the wound area in reference to the bilateral cell sheets. Readers can choose the one that best represents their needs.
This scratch wound migration/invasion assay can be convenient, but it requires the 96-pin scratch tool, designated plates, and the live-cell imager with the integrated migration/invasion module to reproduce the typical results presented in this protocol, and these can be expensive. Cost-effective substitutions such as a flat bottom 96-well multiwell plate can be used and can generate decent scratch wounds (data not shown). However, the performance of the scratch tool was not as good as when the designated plate was used, and some wounds could not be picked up by the scanning mode (Figure 8), thus unable to correctly define the initial wound area. Additionally, the scratch tool can be used to create scratch wounds, with analysis either manually, by image processing programs, or using other end-point visualization and analysis tools. They provide more flexibility to locate the initial wounds, and therefore are not limited to the designated plates. On the other hand, the migration/invasion assay combined with live-cell imaging in the current protocol is less time-consuming and less error-prone. This could be particularly useful for experimental consistency and increased efficiency.
In conclusion, this migration/invasion assay is quick, easy and robust. Users can choose from our protocol and others based on their need and budget.

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.

<table><tbody><tr><td>0.5% trypsin-EDTA solution (10x)</td><td>ThermoFisher Scientific</td><td>30028-02</td><td>Dilute to 2x in DPBS</td></tr><tr><td>Countess II FL Automated Cell Counter</td><td>ThermoFisher Scientific</td><td>AMQAX1000</td><td>Automated cell counter</td></tr><tr><td>Detergent 1 (Alconox)</td><td>Sigma-Aldrich</td><td>242985</td><td>0.5% working concentration</td></tr><tr><td>Detergent 2 (Virkon S)</td><td>VetProduct DIRECT</td><td></td><td>1% working concentration</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium, no phenol-red</td><td>ThermoFisher Scientific</td><td>21063-045</td><td>Supplimented with 10% FBS, 200 mM L-glutamine, 2 &amp;mu;g/ml insulin</td></tr><tr><td>ECM Gel (matrigel)</td><td>Sigma-Aldrich</td><td>E6909</td><td>Growth-factor reduced, phenol red free</td></tr><tr><td>Essen ImageLock 96-well plate, flat bottom</td><td>Essen</td><td>4379</td><td></td></tr><tr><td>EVE Counting slides</td><td>BioTools</td><td>EVS-50</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Bovogen Biologicals</td><td>SFBS-F-500ml</td><td></td></tr><tr><td>IncuCyte 96-well scratch wound cell invasion accessories</td><td>Essen</td><td>4444</td><td>Including CoolBox, 2x CoolSink</td></tr><tr><td>IncuCyte Cell migration kit</td><td>Essen</td><td>4493</td><td>Including the 96-well pin block, 2x wash boats and the software</td></tr><tr><td>IncuCyte ZOOM</td><td>Essen</td><td></td><td>Live cell analysis system</td></tr><tr><td>Insulin solution human</td><td>Sigma-Aldrich</td><td>19278-5ML</td><td></td></tr><tr><td>L-glutamine solution (100x)</td><td>ThermoFisher Scientific</td><td>25030-091</td><td></td></tr><tr><td>Tissue culture flask, 75 cm&lt;sup&gt;2&lt;/sup&gt; growth area</td><td>Greiner Bio-One</td><td>658175</td><td></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 wounds in the designated plate and the live-cell imager is designed to take real-time high-resolution images with the scratch wounds in the middle. The two breast cancer cell lines used in the current protocol are ZR75-1 and MDA-MB-231, categorized as luminal B and triple negative subtypes respectively9. The scratch wounds generated by the 96-pin scratch tool are commonly between 700-900 &#181;m but may vary among different cell lines (Figure 2).
For the migration assay, three metrics are provided: (1) Wound width (&#181;m) is the average distance between cell sheets beside the wound (Figure 3). (2) Wound confluence (%) is the confluence within the wounding area (Figure 3). The ideal initial wound confluence should be close to 0%. (3) Relative wound density (%) is a background-subtracted algorithm [%Relative wound density = 100 * (w(t) - w(0)) / (c(t) - w(0)), t = at time t, w = density of wound region, c = density of cell region], measuring the density of the wound region relative to the density of the cell region.
Three eligible metrics can be achieved based on the needs. The relative wound density and wound confluence imply the speed of cells occupying the scratch wound area, and they almost overlap in both cell lines. But the two cell lines showed very different wound healing abilities, where at the completion (circa 50 h) of the wound healing process of MDA-MB-231 cells, ZR75-1 cells had about 50% wound coverage (the two metrics above) and over 400 &#181;m remaining wound width, indicating a lower migration ability of the ZR75-1 cells (Figure 4).
The migrating behaviors of the two cell types were different and can be compared based on the recorded images. Lamellipodia (blebbing protrusions) were observed in MDA-MB-231 cells at the migration front at the beginning of migration, which was a typical sign of cytoskeleton rearrangement, directing cell movement toward the cell-free region10 (Figure 5). ZR75-1 cells showed no sign of cell migration at the same time-point (Figure 5).
The metric Relative Wound Density is recommended by the manufacturer for cell invasion, as it is a 3D assay, and confluence-based analysis is not applicable. Within 50 h, the relative wound density of MDA-MB-231 cells was saturated (100%), whereas the relative wound density of ZR75-1 cells did not change over time, indicating the highly invasive phenotype of MDA-MB-231 cells and the non-invasiveness of ZR75-1 cells (Figure 6).
MDA-MB-231 cells also behaved differently while invading through ECM gel (Figure 7). Compared to cells with bleb at the migration front, invading cells showed an elongated morphology with leading protrusions.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59042/59042fig1.jpg" /> 
Figure 1: Workflow of migration and invasion assay in the current protocol. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59042/59042fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59042/59042fig2.jpg" /> 
Figure 2: Performance of the scratch tool on breast cancer cell lines ZR75-1 and MDA-MB-231 (upper panel) with the measured initial wound width and initial wound confluence (lower panel). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59042/59042fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59042/59042fig3.jpg" /> 
Figure 3: Migration assay illustrated by HD phase contrast images and blended mode with masks in MDA-MB-231. Blue, scratch wound; yellow, cells surrounding the scratch wound; pink, cells that moved into the scratch wound at 24 h after scratch. Scale bars = 300 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59042/59042fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59042/59042fig4.jpg" /> 
Figure 4: Comparison of three algorithms (wound width, relative wound density and wound confluence of breast cancer cell lines ZR75-1 (left) and MDA-MB-231 (right). All experiments represent the mean of three independent experiments, &#177; the S.D. Scale bars = 300 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59042/59042fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59042/59042fig5.jpg" /> 
Figure 5: Migration front of ZR75-1 and MDA-MB-231 cells 4 h after scratch. Arrows, lamellipodia at the migration front. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59042/59042fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59042/59042fig6.jpg" /> 
Figure 6: Quantification of invasion using metric Relative Wound Density (%) of breast cancer cell line ZR75-1 (left) and MDA-MB-231 (right). All experiments represent the mean of three independent experiments, &#177; the S.D. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59042/59042fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59042/59042fig7.jpg" /> 
Figure 7: Different cellular characteristics of MDA-MB-231 cells in migration (left) and invasion (right). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59042/59042fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/59042/59042fig8.jpg" /> 
Figure 8: An example of a scratch wound that cannot be analyzed by the integrated migration module from a 96-well plate. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59042/59042fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

a-simple-migrationinvasion-workflow-using-an-automated-live-cell

Nanyang Technological University

Research

JoVE Journal

Cancer Research

7.7K Views.  Hunter Medical Research Institute. 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.

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

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Immunology and Infection

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Bioengineering

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

Medicine

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell&#8217;s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using three-dimensional (3D) cell-based invasion and migration assays. In this study, we have optimized these 3D assays for live-cell immunocytochemistry. An Antibody Labeling Reagent was used to detect in real-time the expression of the adhesion molecule CD44, on the plasma membrane of migrating and invading cells of a DMG H3K27M primary patient-derived cell line. CD44 is associated with cancer stem cell phenotype and tumor cell migration and invasion and is involved in the direct interactions with the central nervous system (CNS) extracellular matrix. Neurospheres (NS) from the DMG H3K27M cell line were embedded into the basal membrane matrix (BMM) or placed onto a thin coating layer of BMM, in the presence of an anti-CD44 antibody in conjunction with the antibody labeling reagent (ALR). The live-3D-cell immunocytochemistry image analysis was performed on a live-cell analysis instrument to quantitatively measure the overall CD44 expression, specifically on the migrating and invading cells. The method also allows visualizing in real-time the intermittent expression of CD44 on the plasma membrane of migrating and invading cells. Moreover, the assay also provided new insights into the potential role of CD44 in the mesenchymal to amoeboid transition in DMG H3K27M cells.

This study presents a protocol of live-3D-cell immunocytochemistry applied to a pediatric diffuse midline glioma cell line, useful to study in real-time the expression of proteins on the plasma membrane during dynamic processes like 3D cell invasion and migration.

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Biology

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

Imaging Neutrophil Migration in the Mouse Skin to Investigate Subcellular Membrane Remodeling Under Physiological Conditions

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first immune cells to reach the site of inflammation and to participate in the innate immune response during infection or tissue damage. So far, neutrophil migration has been successfully visualized using various in vitro experimental systems based on uniform stimulation, or confined migration under agarose, or micro-fluidic channels. However, these models do not recapitulate the complex microenvironment that neutrophils encounter in vivo. The development of multiphoton microscopy (MPM)-based techniques, such as intravital subcellular microscopy (ISMic), offer a unique tool to visualize and investigate neutrophil dynamics at subcellular resolutions under physiological conditions. In particular, the ear of a live anesthetized mouse provides an experimental advantage to follow neutrophil interstitial migration in real-time due to its ease of accessibility and lack of surgical exposure. ISMic provides the optical resolution, speed, and depth of acquisition necessary to track both cellular and, more importantly, subcellular processes in 3D over time (4D). Moreover, multi-modal imaging of the interstitial microenvironment (i.e., blood vessels, resident cells, extracellular matrix) can be readily accomplished using a combination of transgenic mice expressing select fluorescent markers, exogenous labeling via fluorescent probes, tissue intrinsic fluorescence, and second/third harmonic generated signals. This protocol describes 1) the preparation of neutrophils for adoptive transfer into the mouse ear, 2) different settings for optimal sub-cellular imaging, 3) strategies to minimize motion artifacts while maintaining a physiological response, 4) examples of membrane remodeling observed in neutrophils using ISMic, and 5) a workflow for the quantitative analysis of membrane remodeling in migrating neutrophils in vivo.

Neutrophil migration relies on the rapid and continuous remodeling of the plasma membrane in response to chemoattractant and its interactions with the extracellular microenvironment. Described herein is a procedure based on Intravital Subcellular Microscopy to investigate the dynamic of membrane remodeling in neutrophils injected in the ear of anesthetized mice.

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first ...

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

Summary

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Chapters in this video

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

Modeling and Imaging 3-Dimensional Collective Cell Invasion

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Imaging Neutrophil Migration in the Mouse Skin to Investigate Subcellular Membrane Remodeling Under Physiological Conditions