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 ...

<ol>
	<li>Enyedi, B., Niethammer, P. <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+epithelial+wound+detection.">Mechanisms of epithelial wound detection.</a> Trends Cell Biol. 25, 398-407 (2015).</li><li>Kujath, P., Kujath, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complicated+skin,+skin+structure+and+soft+tissue+infections+-+are+we+threatened+by+multi-resistant+pathogens?.">Complicated skin, skin structure and soft tissue infections - are we threatened by multi-resistant pathogens?.</a> Eur J Med Res. 15, 544-553 (2010).</li><li>Moreau-Marquis, S., Stanton, B. A., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+biofilm+formation+in+the+cystic+fibrosis+airway.">Pseudomonas aeruginosa biofilm formation in the cystic fibrosis airway.</a> Pulm Pharmacol Ther. 21, 595-599 (2008).</li><li>Chiappini, E., Taccetti, G., de Martino, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+lung+infections+in+cystic+fibrosis+patients:+an+update.">Bacterial lung infections in cystic fibrosis patients: an update.</a> Pediatr Infect Dis J. 33, 653-654 (2014).</li><li>Mangoni, M. L., McDermott, A. M., Zasloff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides+and+wound+healing:+biological+and+therapeutic+considerations.">Antimicrobial peptides and wound healing: biological and therapeutic considerations.</a> Exp Dermatol. 25, 167-173 (2016).</li><li>Lau, K., Paus, R., Tiede, S., Day, P., Bayat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+role+of+stem+cells+in+cutaneous+wound+healing.">Exploring the role of stem cells in cutaneous wound healing.</a> Exp Dermatol. 18, 921-933 (2009).</li><li>Hu, M. 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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPed+up+immunity:+how+antimicrobial+peptides+have+multiple+roles+in+immune+defense.">AMPed up immunity: how antimicrobial peptides have multiple roles in immune defense.</a> Trends Immunol. 30, 131-141 (2009).</li><li>Zasloff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides+of+multicellular+organisms.">Antimicrobial peptides of multicellular organisms.</a> Nature. 415, 389-395 (2002).</li><li>Hulkower, K. I., Herber, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+and+invasion+assays+as+tools+for+drug+discovery.">Cell migration and invasion assays as tools for drug discovery.</a> Pharmaceutics. 3, 107-124 (2011).</li><li>Di Grazia, 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=The+frog+skin-derived+antimicrobial+peptide+esculentin-1a(1-21)NH2+promotes+the+migration+of+human+HaCaT+keratinocytes+in+an+EGF+receptor-dependent+manner:+a+novel+promoter+of+human+skin+wound+healing?.">The frog skin-derived antimicrobial peptide esculentin-1a(1-21)NH2 promotes the migration of human HaCaT keratinocytes in an EGF receptor-dependent manner: a novel promoter of human skin wound healing?.</a> PLoS One. 10, e0128663 (2015).</li><li>Di Grazia, 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=D-Amino+acids+incorporation+in+the+frog+skin-derived+peptide+esculentin-1a(1-21)NH2+is+beneficial+for+its+multiple+functions.">D-Amino acids incorporation in the frog skin-derived peptide esculentin-1a(1-21)NH2 is beneficial for its multiple functions.</a> Amino Acids. 47, 2505-2519 (2015).</li><li>Cappiello, F., 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=Esculentin-1a-derived+peptides+promote+clearance+of+Pseudomonas+aeruginosa+internalized+in+bronchial+cells+of+cystic+fibrosis+patients+and+lung+cell+migration:+biochemical+properties+and+a+plausible+mode+of+action.">Esculentin-1a-derived peptides promote clearance of Pseudomonas aeruginosa internalized in bronchial cells of cystic fibrosis patients and lung cell migration: biochemical properties and a plausible mode of action.</a> Antimicrob Agents Chemother. 60, 7252-7262 (2016).</li><li>Bebok, Z., 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=Failure+of+cAMP+agonists+to+activate+rescued+deltaF508+CFTR+in+CFBE41o-+airway+epithelial+monolayers.">Failure of cAMP agonists to activate rescued deltaF508 CFTR in CFBE41o- airway epithelial monolayers.</a> J Physiol. 569, 601-615 (2005).</li><li>Trinh, N. T., 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=Improvement+of+defective+cystic+fibrosis+airway+epithelial+wound+repair+after+CFTR+rescue.">Improvement of defective cystic fibrosis airway epithelial wound repair after CFTR rescue.</a> Eur Respir J. 40, 1390-1400 (2012).</li><li>Gan, H. K., 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+epidermal+growth+factor+receptor+(EGFR)+tyrosine+kinase+inhibitor+AG1478+increases+the+formation+of+inactive+untethered+EGFR+dimers.+Implications+for+combination+therapy+with+monoclonal+antibody+806.">The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 increases the formation of inactive untethered EGFR dimers. 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. 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=The+antimicrobial+peptide+LL-37+activates+innate+immunity+at+the+airway+epithelial+surface+by+transactivation+of+the+epidermal+growth+factor+receptor.">The antimicrobial peptide LL-37 activates innate immunity at the airway epithelial surface by transactivation of the epidermal growth factor receptor.</a> J Immunol. 171, 6690-6696 (2003).</li><li>Di Grazia, A., Luca, V., Segev-Zarko, L. A., Shai, Y., Mangoni, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporins+A+and+B+stimulate+migration+of+HaCaT+keratinocytes+and+kill+intracellular+Staphylococcus+aureus.">Temporins A and B stimulate migration of HaCaT keratinocytes and kill intracellular Staphylococcus aureus.</a> Antimicrob Agents Chemother. 58, 2520-2527 (2014).</li></ol>

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 ce...

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 border="0" cellpadding="0" cellspacing="0" width="852">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Minimal essential medium (MEM)&#160;</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECB2071L</td>
			<td style="width:256px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Glutamine</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECB3000D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Heat inactivated Fetal Bovine Serum (FBS)</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECS0180DH&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Penicillin and Streptomycin</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECB3001D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Puromycin</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Trypsin/EDTA 1X in PBS</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECB3052D</td>
			<td>Warm at room temperature before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">DPBS without calcium and magnesium (CMF-PBS)</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">DPBS with calcium and magnesium (PBS)</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8662</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Ibidi Culture-Insert 2 well</td>
			<td style="width:179px;">Ibidi&#160;</td>
			<td style="width:119px;">80209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Wimasis Image Analysis</td>
			<td style="width:179px;">Ibidi&#160;</td>
			<td style="width:119px;">30002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PRISM software&#160;</td>
			<td>GraphPad</td>
			<td style="width:119px;">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 containi...

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 ...

The goal of this procedure is to show how to evaluate the ability of some immunomodulatory molecules, such antimicropeptides, to stimulate cell migration, which is a rate-limiting event during the wound-healing process. Generally speaking, the basic procedure to study cell migration in vitro involves the creation of cell monolayer, the production of pseudo-wound, the capture of images at different time intervals until wound closure is reached, and analysis of the image sequence to quantify cell migration. Now, the advantage of this method over the classic assay, which is based on a manually made scratch, is due to the usage of special silicone culture inserts to create the pseudo-wound field, which is completely free of cells and with a very well-defined width, 500 micrometer.In addition, thanks to a web-based, automated image analysis program, it is possible to rapidly receive within minutes quantitative data on the speed of wound closure and cell migration. So overall, this is a reliable experimental technique that can be applied to any kind of adherent cells with very high reproducibility of data. Here, it is provided, as an example, the effect of a frog skin antimicropeptide on the migration of bronchial epithelial cells and how pretreatment of these cells with specific inhibitors can give information on the molecular mechanism underlying such events.More precisely, it will be shown how the peptide-induced cell migration involves activation of epidermal growth factor receptor. To begin experiment, remove the flask containing cells from the cell culture incubator, and check the circumference on an inverted microscope equipped with an automatic image acquisition system. If 90%of complete confluence is reached, move to a biosafety hood, and carefully aspirate the culture medium using a 10-milliliter plastic pipette under sterile conditions and gently discard the medium into waste bottle.Wash the cells with six milliliter PBS without calcium and magnesium, that is, CMF. Gently rock the flask manually and discard CMF. Then, add other 10 milliliter CMF to further remove cell debris and non-attached cells.At this point, close the flask, and put it back into the cell culture incubator for 10 minutes. Take out the flask, discard CMF, and gently add two milliliter trypsin-EDTA to detach cells from the plastic surface. Gently rock the flask manually, allowing the solution to completely cover the cells, and incubate the flask for additional 10 minutes.Afterwards, remove the flask from the incubator, and check whether the trypsin addition process is complete. If this is the case, the cells will be in suspension and will appear rounded under the microscope. To inactivate trypsin, add 10 milliliter culture media supplemented with 10%heat-inactivated bovine serum, and carefully wash the flask.Collect the cell suspension, transfer this volume into a conical 50-milliliter tube, and centrifuge the sample. After centrifugation, check the pellet formation, aspirate the supernatant, and resuspend the cell pellet in six milliliter culture medium supplemented with serum. Pipette the cell suspension up and down for several times to break up any clumps formation.This process may take some minutes. Afterwards, take out 10 microliter with a micropipette tip, and inject the volume under the cover glass of a Burker or Neubauer chamber and count the cell number. In each well of a 12-well plate, put the culture insert with the help of sterile tweezers.Each insert is provided of two compartments separated by a 500-micrometer wall, and there's a sticky underside to allow adhesion. However, by means of tweezers, apply some pressure along the insert edges to fix them to the surface of the plate. Fill each compartment of the insert with 70 microliter of cell suspension containing about 3.5 by 10 to fourth cells per chamber.Importantly, the concentration of cells applied into each compartment depends on the type of cells. It is recommended to use a cell density that leads to complete confluence within 24 hours. Under the microscope at four-fold magnification, check that cells are not leaking from the insert compartments, and then incubate the plate for 24 hours.After incubation, gently remove the inserts with sterile tweezers without breaking the cell monolayers, and transfer the insert onto absorbent paper. Wash the cells by adding one milliliter medium per well. Be careful not to add the medium directly on top of cell monolayers, to avoid their disruption.Close the plate, and gently rock it manually. Aspirate the medium, and replace it with one milliliter of fresh medium per well. Visualize the cell-free gaps under the microscope, and acquire images at time zero.Withdraw the media from the wells, and add PBS supplemented with calcium and magnesium. Shake the plate, remove PBS, and finally add the test compounds to the wells. For untreated control samples, one millimeter medium is used.Incubate the plate. For each sample, observe the cell migration under the microscope at four-fold magnification, and acquire images after 15, 20, and 24 hours incubation. During this step, try to capture images in the same areas as for time zero.The choice of time intervals at which images are captured depends on the migration speed of the selected cells. When the experiment is finished, select images for each sample at the different time intervals, and create a zip file. It is important to have at least three replicates for each sample at each time interval.Open the automated Image Analysis Wimasis program, and upload the zip file using the lowest magnification. The program will automatically provide by email a summary file containing the experimental data of cell-covered area and scratch areas as percentages of each single sample, for example, samples 1a and 2a, at the selected time points. Calculate a mean value of all samples at time zero.Normalize all data with respect to the mean value at time zero. Calculate a mean value of all samples at each time point and the relative standard error. By using two-way ANOVA test, perform statistical analysis.The obtained data can be plot into a graph as a histogram showing the percentage of cell-covered area at different time points for different sample groups. These histograms show the wound closure obtained after 15, 20, and 24 hours treatment of bronchial cells with different concentrations of the antimicropeptide esculentin 1-21-1c or esculentin 1-21 in comparison to the control. As pointed out by the histograms and some representative micrographs, esculentin 1-21-1c is more efficient in promoting re-epithelialization of pseudo-wound in bronchial cells induced in the almost-complete closure of the wound field within approximately 20 hours at one micromolar, while a higher concentration is needed for esculentin 1-21, that is, 10 micromolar.To study the role of some receptors, such as epidermal growth factor receptor, on cell migration, pretreat the cell monolayers with a specific inhibitor, for example, the compound AG1478. To this purpose, before removing the insert, aspirate the medium in each compartment, wash it once, and add 70 microliter of media supplemented with five micromolar AG1478. Incubate the plate for 30 minutes.Then, remove the insert with tweezers, and proceed as described earlier. These are the final data showing that the peptide-induced cell migration involves activation of epidermal growth factor receptor. Indeed, after pretreatment with inhibitor AG1478, the percentage of cell-covered area is significantly reduced at all time intervals compared to the results found when the cells are not pre-incubated with AG1478 before adding the peptide.In conclusion, the described method has a high potential to significantly improve the understanding on the migratory feature of different types of cells, as well as on the selection of the most promising wound-healing promoters and/or inhibitors, within a short time and with a high accuracy.

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Immunology and Infection

21.3K vistas.  Sapienza University of Rome. The goal of this procedure is to show how to evaluate the ability of some immunomodulatory molecules, such antimicropeptides, to stimulate cell migration, which is a rate-limiting event during the wound-healing process. Generally speaking, the basic procedure to study cell migration in vitro involves the creation of cell monolayer, the production of pseudo-wound, the capture of images at different time intervals until wound closure is reached, and analysis of the image sequence to quantify ce...