This work was supported by funding from Sapienza University of Rome and the Italian Cystic Fibrosis Research Foundation (Project FFC#11/2014 adopted by FFC Delegations from Siena, Sondrio Valchiavenna, Cerea Il Sorriso di Jenny, and Pavia). Part of this work was also supported by FILAS Grant Prot. FILAS-RU-2014-1020. 
We are grateful to Dr. Loretta Ferrera (U.O.C. Genetica Medica, Istituto Giannina Gaslini, Genova, Italy) for providing the bronchial epithelial cells.

1. Cell Preparation
<ol>
	<li>Seed 2.5x106 cells in 10 mL of Minimum Essential Medium (MEM) supplemented with 2 mM glutamine (MEMg), plus 10% fetal bovine serum (FBS), antibiotics (0.1 mg/mL of penicillin and streptomycin), and puromycin (0.5 &#181;g/mL for selection and maintenance of the cell line) in a T75 flask. Incubate the flask at 37 &#176;C and 5% CO2 for 2 days. Before starting the experiment, check the confluence of cells under an inverted microscope. 
	NOTE: The cells used for the experiment are immortalized human bronchial epithelial cells transduced with a lentiviral vector conferring resistance to puromycin. They stably express a functional CFTR14,15.</li>
	<li>Once the cells&#39; confluence has reached 90-100%, aspirate the medium from the flask and discard it into a waste bottle under a biological safety cabinet class II. Wash the cells with 6 mL of phosphate buffered saline without calcium and magnesium (CMF-PBS). Gently rock the flask manually and discard CMF-PBS. 
	NOTE: Be careful not to touch the cell monolayer with the pipette.</li>
	<li>Add 10 mL of CMF-PBS and incubate the flask at 37 &#176;C and 5% CO2 for 10 min.</li>
	<li>Aspirate CMF-PBS and discard it. Then add 2 mL of trypsin/EDTA to the flask.</li>
	<li>Gently rock the flask, allowing the solution to completely coat the cells, and incubate the flask at 37 &#176;C and 5% CO2 for 10 min until the cells are visibly detached under a microscope. 
	NOTE: At the end of the incubation time, the cells should appear rounded and not attached to the plastic surface. If the cells are not well detached, manual agitation may be necessary.</li>
	<li>Add 10 mL of MEMg plus 10% FBS to inactivate trypsin and collect the cells by washing the bottom of the flask. Transfer the volume into a conical 50 mL tube.</li>
	<li>Centrifuge the tube for 5 min at 80 x g.</li>
	<li>Aspirate the supernatant and re-suspend the cells in 6 mL of MEMg plus 10% FBS. Pipette repeatedly to break up any clumps.</li>
	<li>Take out 10 &#181;L of cell suspension with a micropipette and inject the volume under the cover glass previously put over a Burker or Neubauer chamber.</li>
	<li>Count the cell number.</li>
</ol>
2. Cell Seeding in the Culture Inserts
<ol>
	<li>In each well of a 12-well plate, transfer the culture insert with sterile tweezers (Figure 1). Use tweezers to press along the inserts edges in order to fix them to the surface of the plate. 
	NOTE: Inserts have a sticky underside that allows adhesion.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56825/56825fig1.jpg" /> 
Figure 1: Schematic representation of the silicone culture inserts, properly put into wells of a 12-well plate. <a href="//cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56825/56825fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Properly dilute the cell suspension in MEMg plus 10% FBS. Fill each compartment of the insert with 70 &#181;L of cell suspension (about 3.5x104 cells/chamber). 
	NOTE: The density of cells applied in each compartment depends on the type of cells. It is recommended to use a cell density that leads to complete confluence within 24 h.</li>
	<li>Under the microscope, check that cells are not leaking from the insert compartments and incubate the 12-well plate for 24 h at 37 &#176;C and 5% CO2.</li>
</ol>
3. Pseudo-Wound Healing Assay
<ol>
	<li>After incubation, visualize the cells under the inverted microscope to verify that confluent cell monolayers have been formed.</li>
	<li>Re-suspend the test compounds (e.g., AMPs) in 1 mL of MEMg. 
	NOTE: Prepare fresh AMPs dilutions starting from the stock solution stored at -20 &#176;C.</li>
	<li>Gently remove the inserts by sterile tweezers. Be careful not to break the cell monolayers. Transfer the inserts onto absorbent paper. 
	NOTE:&#160;To re-use the same inserts, sterilize them in 70% ethanol for at least 3 h. It is recommended to throw them away afterwards.</li>
	<li>To remove non-adherent cells, add 1 mL of MEMg per well using a micropipette. Close the plate and gently rock it. 
	NOTE: Do not add medium directly on top of cell monolayers to avoid their disruption.</li>
	<li>Aspirate the medium and replace it with 1 mL of MEMg per well. Close the plate and visualize the cell-free gaps (created by the inserts) under the inverted microscope at 4X magnification, equipped with a video-camera. Acquire images at time zero (T0) and save them in a .jpg format.</li>
	<li>Remove the medium from the wells, wash them with 1 mL of PBS, and discard it.</li>
	<li>Add the test compounds (prepared at point 3.2) to the wells. For untreated control samples, add 1 mL of MEMg. Incubate the plate at 37 &#176;C and 5% CO2.</li>
	<li>After 15, 20, and 24 h treatment, observe the cell migration under the microscope at 4X magnification and acquire images. 
	NOTE: During this step, try to capture images in the same areas as for T0. The choice of time intervals at which images are captured depends on the cell migration speed.</li>
	<li>To study the effect of some selective inhibitors, i.e. AG1478, on cell migration, aspirate the medium from each insert compartment before removing inserts.
	<ol>
		<li>Wash each compartment with fresh MEMg and fill it with 70 &#181;L of MEMg supplemented with AG1478.</li>
		<li>After 30 min of incubation at 37 &#176;C and 5% CO2, proceed as described from point 3.3. 
		NOTE: During the washing step and pretreatment of cell monolayers with the specific inhibitors, be careful not to remove the inserts.</li>
	</ol>
	</li>
</ol>
4. Image Analysis
<ol>
	<li>Upon completion of the experiment, select images of the most representative samples of the various experimental groups, and create a zip file containing single images at T0, T15, T20, and T24 h. 
	NOTE: Single images are taken at the selected time intervals for all samples. Run triplicates for each experiment, which is repeated at least three times. At the end, for all experimental groups, a minimum of 3 images (&#34;a&#34;, &#34;b&#34;, &#34;c&#34;, etc., deriving from each independent experiment) at each time point are analyzed.</li>
	<li>Upload the zip file into the image analysis software which automatically provides by e-mail a spreadsheet summary file containing the experimental data of cell-covered and scratch areas (as percentage) at the selected time points. 
	NOTE: The recognition of the leading edge and the gap area is largely based on edge detection method (aimed at identifying points at which the image brightness sharply changes).</li>
	<li>Save the data and collect them. Normalize all data with respect to the mean value at time zero. Calculate the average value of the normalized data of all replicates at each time point and the relative standard error (SEM). By using two-way analysis of variance (ANOVA), perform statistical analysis with a proper statistics software. Differences between peptide-treated groups and control groups at different time intervals are considered to be statistically signi&#64257;cant for a p&#60;0.05.</li>
	<li>Plot the obtained data into a graph as a histogram, which shows the percentage of cell-covered area of all sample groups versus the selected time intervals.</li>
</ol>

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S., 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=Tissue+engineering+and+regenerative+repair+in+wound+healing.">Tissue engineering and regenerative repair in wound healing.</a> Ann Biomed Eng. 42, 1494-1507 (2014).</li><li>Ramot, Y., 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=The+role+of+PPARgamma-mediated+signalling+in+skin+biology+and+pathology:+new+targets+and+opportunities+for+clinical+dermatology.">The role of PPARgamma-mediated signalling in skin biology and pathology: new targets and opportunities for clinical dermatology.</a> Exp Dermatol. 24, 245-251 (2015).</li><li>Lai, Y., Gallo, R. 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Implications for combination therapy with monoclonal antibody 806.</a> J Biol Chem. 282, 2840-2850 (2007).</li><li>Tokumaru, S., 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=Induction+of+keratinocyte+migration+via+transactivation+of+the+epidermal+growth+factor+receptor+by+the+antimicrobial+peptide+LL-37.">Induction of keratinocyte migration via transactivation of the epidermal growth factor receptor by the antimicrobial peptide LL-37.</a> J Immunol. 175, 4662-4668 (2005).</li><li>Tjabringa, G. 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The authors have nothing to disclose

Cell migration is an essential process in many physiological and pathological events including wound healing, embryonic development, and cancer metastasis. The basic procedure to study cell migration in vitro involves: (i) the creation of a cell monolayer, (ii) the production of a pseudo-wound in the confluent layer of cells, (iii) the capture of images at different time intervals until wound closure is reached, and (iv) the analysis of the image sequence in order to quantify the migration speed of the chosen cells.
Our results have shown that the application of culture inserts is an objective and reliable experimental technique for quantitative evaluation of cell migration characteristics. This assay can be performed by any research group with basic laboratory equipment. However, some critical steps of the protocol should be considered to achieve reproducibility. For example, it is important to define, by pilot experiments, the exact number of cells to seed in each compartment in order to get the same extent of confluence in all individual samples. To this aim, having cells with a synchronized cycle phase and avoiding cell clumps before seeding is critical. A possible way to minimize clumping is a gentle passage of cell suspension through a syringe needle. However, increasing the samples size will significantly contribute to decrease variability among the cells.
In addition, the creation of a gap with a well-defined width in all samples is crucial. For this purpose, silicone inserts should not be re-used more than twice. Furthermore, the growth of cells under the silicone separation wall should be avoided. To prevent this trouble, soft pressure on top of the insert, once placed into the dish plate/well, can be manually applied by means of sterile tweezers to increase the adhesion of the insert to the plastic. Cautious removal of the insert will also hamper enlargement of the cell-free surface with disruption of the cell monolayer.
However, once the insert is correctly dislodged and a pseudo-wound is perfectly produced, some cell lines can detach from the plastic surface, especially if they are incubated in media without serum or supplements such as vitamins, amino acids, and growth factors. The absence of these factors in a cell culture medium can be dictated by the fact that they can interfere with the wound-healing promoting activity of test compounds. To circumvent this problem, rational choice of culture medium composition is highly recommended.
Another critical step to consider before setting up this wound healing procedure is the different migration speed that exists among different cell types. Also in this case, preliminary experiments need to be scheduled with the purpose of determining the correct time intervals at which images have to be captured. It is also necessary to consider that one limitation associated with this method is that such culture inserts cannot be employed for studying the migration of cells which grow in suspension (e.g., polymorphonuclear leukocytes).
Despite wound-healing assays performed with the silicone culture inserts are more expensive than the classical scratch assay, they allow a quick and accurate reproduction of data on cell migration activity. Importantly, once the system is set-up, it can be used to expand knowledge on the molecular mechanism underlying the wound healing activity stimulated by either endogenous or exogenous factors. To this end, pretreatment of cell monolayers with specific molecules (e.g., inhibitors) can be carried out before creating the pseudo-wound. An example is given by pretreatment of cells with (i) the cell proliferation blocker mitomycin C to explore whether the closure of the &#34;wounded&#34; field induced by AMPs is influenced by cell division rate20 or (ii) metalloprotease inhibitors to verify the contribution of metalloproteases in the activation of EGFR-mediated signaling pathway12.
Importantly, during the wound closure process, samples may be fixed and treated with appropriate stains for cytoskeleton and nuclei detection (phalloidin and DAPI, respectively) to visualize changes in the morphological aspect of cells (e.g., the formation of lamellipodia at the front edge) by fluorescent/confocal microscopy. Moreover, treatment of cells with a fluorescent-labeled AMP may enable the visualization of its cellular distribution (membrane surface or intracellular/intranuclear localization) at different times, since the beginning of the cell migratory activity. Furthermore, these inserts may be exploited for analyzing wound healing activity on polarized and differentiated epithelial cells (e.g., primary bronchial cells maintained at the air-liquid interface), after having placed them on suitable transwell permeable supports.
In conclusion, the described method has substantial potential to significantly improve the understanding on the migratory features of different types of adherent animal cells/epithelial tissues, as well as on the selection of the most promising wound-healing promoters and/or inhibitors, within a short time and with high accuracy.

It is largely known that wound healing in animals is a fundamental process to re-establish the integrity and normal function of tissue layers after injury1. Despite epithelial surfaces exposed to the external environment (e.g., the skin, respiratory, and gastrointestinal tracts) form&#160;a protective barrier from physical and chemical insults, the formation of wounds can easily occur, especially after surgery or microbial infections2. As an example, colonization of lung tissue by the opportunistic bacterial pathogen Pseudomonas aeruginosa, especially in cystic fibrosis (CF) sufferers, leads to damage of the airways epithelium with consequent respiratory failure3,4. Wound healing is a complex host repair mechanism to restore the normal architecture of an injured tissue5. It is characterized by initial inflammation, followed by a regeneration period encompassing epithelialization, angiogenesis, and tissue remodeling with collagen production and cell differentiation6,7,8. To ensure epithelial integrity and to control microbial proliferation, all living organisms produce defense molecules, including antimicrobial peptides (AMPs)9,10. The wound healing process is very difficult to simulate in vitro due to the lack of cell debris and complex interactions among different cell types. However, the in vitro ability of a peptide to accelerate the closure of a pseudo-wound by stimulating migration of epithelial cells is indicative of its ability to heal a compromised epithelium. Indeed, cell migration is a rate-limiting event in wound healing, and studying factors that can affect cell migration will help to target therapies for improved wound healing.
Here, a highly reproducible experimental assay is provided based on special silicone culture inserts to evaluate cell migration in vitro. It is based on the creation of a 500 &#956;m gap (pseudo-wound) on a confluent cell monolayer. The cells at the edge of the artificial &#34;wounded&#34; field will start migrating into the cell-free area, forming new cell-cell contacts. The culture insert represents a new tool for fast wound healing experiments. Two reservoirs separated by a 500 &#956;m wall are provided, and they can be properly placed into a 3-cm dish plate or in the well of a 12-well plate. Filling each compartment of the insert with a cell suspension allows cells to grow in each designated area until confluence, while removal of the insert will engender a clean cell-free gap of approximately 500 &#956;m (the same width as the separation wall). A proper cell culture medium supplemented with a test compound can then be added into the dish plate/well. Afterwards, the gap closure can be visualized at different time intervals under an inverted microscope, preferably one equipped with a video-camera for image acquisition. Finally, measurement of changes in the cell-covered area by the web-based automated image analysis program will allow the quantification of the speed of wound closure and cell migration. Overall, this method is a step forward with respect to the classical assay, where a scratch is manually made by incising confluent cell monolayers with a sterile needle or a pipette tip11. Indeed, the last procedure can destroy the plastic bottom of the dish plate/well and the surface coating, creating wrinkles. In addition, the &#34;wounded&#34; area does not have a well-defined width along the entire length of the gap, as this highly depends on the pressure applied by researchers to the needle/tip. Furthermore, the dislodged cells can form clumps of living and dead cells at the edges of the scratch; moreover, the spreading of living cells into the &#34;wounded&#34; area can interfere with the velocity of cell migration, generating non-reproducible results12.
In addition, thanks to a scratch image analysis platform, users can rapidly receive (within minutes) quantitative data on the migratory behavior of the selected cells without the necessity of acquiring additional software. This platform is capable of analyzing phase contrast microscopy images of low (~5X), medium (~10X), and high (~20X) magnification. After uploading a zip file of images (in *.jpg, *.jpeg, *.jp2, *.png, *.gif, *.tiff format) the analysis is automatically conducted to generate a summary file that shows the percentage of both cell-covered areas and scratch areas, as well as the speed of cell migration, at distinct time intervals.
In this work, by using a frog-skin AMP-derivative, i.e. Esc(1-21) and its diastereomer Esc(1-21)-1c13, and a bronchial cell line expressing the functional CF transmembrane conductance regulator (CFTR)14,15, an example of peptide-induced cell migration in comparison with untreated (control) samples is provided. Note that the airway epithelium and CFTR play a crucial role in maintaining lung function and wound repair16. Furthermore, by means of selective inhibitors (e.g., AG1478)17 of the epidermal growth factor receptor (EGFR), evidence that migration of bronchial cells induced by the aforementioned peptides involves activation of EGFR12,18 is reported.
In summary, the goal of this procedure is to show how the usage of such silicone culture inserts represents a fast and easily accessible assay to accurately determine migration of adherent cells (e.g., bronchial epithelial cells) and the molecular mechanisms controlling such events.

<table><tbody><tr><td>Minimum essential medium (MEM)&amp;nbsp;</td><td>Euroclone</td><td>ECB2071L</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Glutamine</td><td>Euroclone</td><td>ECB3000D</td><td></td></tr><tr><td>Heat inactivated Fetal Bovine Serum (FBS)</td><td>Euroclone</td><td>ECS0180DH&amp;nbsp;</td><td></td></tr><tr><td>Penicillin and Streptomycin</td><td>Euroclone</td><td>ECB3001D</td><td></td></tr><tr><td>Puromycin</td><td>Sigma-Aldrich</td><td>P8833</td><td></td></tr><tr><td>Trypsin/EDTA 1X in PBS</td><td>Euroclone</td><td>ECB3052D</td><td>Warm at room temperature before use</td></tr><tr><td>DPBS without calcium and magnesium (CMF-PBS)</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>DPBS with calcium and magnesium (PBS)</td><td>Sigma-Aldrich</td><td>D8662</td><td></td></tr><tr><td>Ibidi Culture-Insert 2 well</td><td>Ibidi&amp;nbsp;</td><td>80209</td><td></td></tr><tr><td>Wimasis Image Analysis</td><td>Ibidi&amp;nbsp;</td><td>30002</td><td></td></tr><tr><td>PRISM software&amp;nbsp;</td><td>GraphPad</td><td>version 6.0</td><td></td></tr></tbody></table>

a novel in vitro wound healing assay to evaluate cell migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

This protocol was used to determine the wound healing effect of Esc(1-21) and Esc(1-21)-1c in terms of cell migration activity induced on bronchial epithelial cells expressing the functional CFTR. In this assay, culture inserts were placed in wells of a 12-well plate, and each compartment was seeded with 35,000 cells in MEMg supplemented with 10% FBS. The cells reached complete confluence within 24 h. Afterwards, a 500 &#956;m gap was generated, and each well was filled with MEMg containing the peptide at different concentrations. The experiment was repeated four times. As indicated in Figure 2, the almost complete closure of the pseudo-wound field produced in the cell monolayer was induced by the two peptides within ~20 h, at optimal concentrations of 1 &#956;M and 10 &#956;M for Esc(1-21)-1c and Esc(1-21), respectively. This indicates that Esc(1-21)-1c is more efficient in promoting re-epithelialization of a pseudo-wound in bronchial cells.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56825/56825fig2v3.jpg" /> 
Figure 2: Effect of esculentin-1a derived AMPs on the closure of a pseudo-wound created in a monolayer of bronchial epithelial cells. Cells were seeded in each compartment of the silicone culture insert and, after its removal, cells were photographed for evidence of cell migration 15, 20, and 24 h after the addition of peptides. The percentage of cell-covered area at each time point was calculated compared to the untreated control cells (Ctrl) and is reported on the y axis. All data are the means from four independent experiments &#177; SEM, and are taken from a previous study14. The levels of statistical significance for tests between control and treated samples are: *p&#60;0.05, **p&#60;0.01, ***p&#60;0.001, and ****p&#60;0.0001. Micrographs showing representative results before (T0) and 20 h after treatment of cells with each peptide at the concentration of 1 &#956;M, in comparison to the control, are also reported. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56825/56825fig2v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To verify whether activation of EGFR played a role in the Esc-induced wound closure, bronchial cells were preincubated for 30 min with 5 &#956;M of AG1478, a selective inhibitor of EGFR, before insert removal. Since the percentage of cell-covered area was significantly reduced at all time intervals compared to the results found when the cells were not pretreated with the inhibitor prior peptide administration (Figure 3), this points out that peptide-induced cell migration involves activation of EGFR. However, further studies will be needed to clarify the molecular events triggered by the Esc-peptides (e.g., direct EGFR activation or trans-activation mediated by metalloproteases, which have been shown to cleave membrane-anchored EGFR ligands19).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56825/56825fig3v3.jpg" /> 
Figure 3: Effect of AG1478 inhibitor on the peptide-induced migration of bronchial epithelial cells. Before insert removal, cells were pre-incubated with 5 &#956;M AG1478 for 30 min and subsequently treated with the peptide alone at the same concentration. All data are the means from four independent experiments &#177; SEM and are taken from our previous work14. The levels of statistical significance for tests between the indicated group samples are: *p&#60;0.05; ****p&#60;0.0001. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56825/56825fig3v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration at JoVE.com

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

21.8K Views.  Sapienza University of Rome. Hier presenteren we een protocol om te evalueren van het effect van peptiden op de migratie van bronchiale epitheliale cellen. Deze methode zorgt voor de snelle en zeer reproduceerbaar accumulatie van kwantitatieve gegevens over de snelheid van cel migratie en wond sluiting.

Video: A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels &#8211; either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention.
We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.

Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

Developmental Biology

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 ...

Cancer Research

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor metastasis. In this assay, cells are grown to form a confluent monolayer and a mechanical wound is created by scratching with a device. Then the migration rate of the cells towards the denuded area can be monitored by imaging. Our 8-channel mechanical wounder is designed to tackle most of the problems associated with the cell migration assay. Firstly, our wounder can be easily sterilized by autoclaving or with common disinfectants. Secondly, the individual adjustable pins allow even contact with the cell culture plate so that sharp and reproducible wounds can be created. Thirdly, the guiding bars on both sides of the wounder ensure consistent wounding position in each well. The use of disposable plastic pipette tips for wounding can further provide better handling of the wounder as well as to minimize cross-contamination. In conclusion, our cell wounder can provide researchers with a user friendly and reproducible device for performing the cell migration assay using the standard 96-well culture plate.

Here, we present a protocol to perform a semi-high throughput cell migration assay on a 96-well cell culture plate. This protocol is a fast, simple and economical method to create consistent scratch wounds on a cell monolayer.

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into changes in clinical standards used for treating wounds and decreasing morbidity and mortality for patients. Wound healing is a complex process that requires strategic cell and tissue interaction and function. One of the many critically important functions of wound healing is individual and collective cellular migration. Upon injury, various cells from the blood, surrounding connective, and epithelial tissues rapidly migrate to the wound site by way of chemical and/or physical stimuli. This migration response can largely dictate the outcomes and success of a healing wound. Understanding this specific cellular function is important for translational medicine that can lead to improved wound healing outcomes. Here, we describe a protocol used to better understand cellular migration as it pertains to wound healing, and how changes to the cellular environment can significantly alter this process. In this example study, dermal fibroblasts were grown in media supplemented with fetal bovine serum (FBS) as monolayer cultures in tissue culture flasks. Cells were aseptically transferred into tissue culture treated 12-well plates and grown to 100% confluence. Upon reaching confluence, the cells in the monolayer were vertically scratched using a p200 pipet tip. Arsenic diluted in culture media supplemented with FBS was added to individual wells at environmentally relevant doses ranging 0.1-10 &#956;M. Images were captured every 4 hours (h) over a 24 h period using an inverted light microscope to observe cellular migration (wound closure). Images were individually analyzed using image analysis software, and percent wound closure was calculated. Results demonstrate that arsenic slows down wound healing. This technique provides a rapid and inexpensive first screen for evaluation of the effects of contaminants on wound healing.

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

This study focuses on an in vitro model of wound healing (scratch assay) as a mechanism for determining how environmental contaminants such as arsenic influence cellular migration. The results demonstrate that this in vitro assay provides rapid and early indications of changes to cellular migration prior to in vivo experimentation.

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into ...

Environment

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Bioengineering

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

Medicine

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Biology

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

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