Name: a novel in vitro wound healing assay to evaluate cell migration
Uploaded: 2018-03-17T13:00:00
Description: Watch this Scientific Journal Video about A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration at JoVE.com

Dette arbejde blev støttet af midler fra Sapienza Universitet i Rom og den italienske cystisk fibrose Research Foundation (projekt FFC #11/2014 vedtaget af FFC delegationer fra Siena, Sondrio Valchiavenna, Cerea Il Sorriso di Jenny og Pavia). En del af dette arbejde blev også støttet af FILAS Grant Prot. FILAS-RU-2014-1020.
Vi er taknemmelige for Dr. Loretta Ferrera (U.O.C. Getica Medica, Istituto Giannina Gaslini, Genova, Italien) til at give de bronchiale epitel celler.

1. celle forberedelse
<ol><li>Seed 2.5x106 celler i 10 mL af Minimum afgørende Medium (MEM) suppleret med 2 mM glutamin (MEMg), plus 10% føtal bovint serum (FBS), antibiotika (0,1 mg/mL af penicillin og streptomycin) og puromycin (0,5 µg/mL for udvælgelse og vedligeholdelse af den celle linje) i en T75 kolbe. Inkuber i kolben på 37 ° C og 5% CO2 i 2 dage. Før du starter eksperimentet, kontrollere sammenløbet af celler i en inverteret mikroskop. Bemærk: De celler, der bruges til eksperi...

<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. 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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>

Celle migration er en væsentlig proces i mange fysiologiske og patologiske arrangementer herunder sårheling, embryonale udvikling og kræft metastaser. Den grundlæggende procedure at studere celle migration i vitro omfatter: (i) oprettelsen af en celle éncellelag, (ii) produktionen af en pseudo-sår i sammenflydende lag af celler, (iii) erobringen af billeder på forskellige tidsintervaller indtil såret lukning er nået , og (iv) analysen af billedsekvens for at kvantificere migration hastigheden af de valg...

Det er i vid udstrækning kendt at sårheling hos dyr er en grundlæggende proces at genskabe integriteten og normal funktion af væv lag efter skade1. Trods epitelial overflader udsat for det ydre miljø (fx, hud, luftveje, og mave skrifter) danner en beskyttende barriere fra fysiske og kemiske fornærmelser, dannelsen af sår kan nemt forekomme, især efter kirurgi eller mikrobielle infektioner2. Som et eksempel, kolonisering af lungevæv ved den opportunistiske bakteriel patogen Pseudomonas aeruginosa, især i cystisk fibrose (CF) syge, fører til skader på luftvejene epitel med deraf følgende respirationssvigt3, 4. Sårheling er en kompleks vært reparation mekanisme til at genoprette den normale arkitektur af en skadet væv5. Det er karakteriseret ved indledende betændelse, efterfulgt af en regenerering periode omfatter epithelialization, angiogenese og væv remodellering med kollagen produktionen og celle differentiering6,7,8 . At sikre epitelial integritet og til at styre mikrobielle spredning, producerer alle levende organismer forsvar molekyler, herunder antimikrobielle peptider (AMP'er)9,10. Sårheling proces er meget vanskeligt at simulere in vitro- på grund af manglende celle debris og komplekse interaktioner blandt forskellige celletyper. Men, in vitro- evne et peptid til at fremskynde lukningen af en pseudo-sår ved at stimulere overflytning af epitelceller er betegnende for dens evne til at helbrede en kompromitteret epitel. Faktisk, celle migration er en trafikhastighedsbegrænsning begivenhed i sårheling, og at studere faktorer, der kan påvirke celle migration vil bidrage til at målrette terapier for forbedret sårheling.
Her, leveres en yderst reproducerbare eksperimentelle assay på basis af særlige silikone kultur skær til at evaluere celle migration in vitro. Det er baseret på oprettelse af en 500 μm gap (pseudo sår) på en sammenflydende celle éncellelag. Celler på kanten af feltet kunstige "sårede" vil begynde at migrere ind i området celle-fri, danner nye celle-celle kontakter. Kultur-Indsæt repræsenterer et nyt værktøj til hurtig sårheling eksperimenter. To bassiner adskilt af en 500 μm væg er leveret, og de kan placeres korrekt, i en 3-cm parabol plade eller i godt af en 12-godt plade. Fylde hvert rum af indsatsen med en cellesuspension tillader celler til at vokse i de enkelte udpegede områder indtil sammenløbet, mens fjernelse af indsatsen vil skabe en ren celle-fri hul ca 500 μm (samme bredde som adskillelsesmuren). En ordentlig cellekulturmedium suppleret med en testforbindelsen kan derefter føjes til fadet plade/godt. Bagefter, gap lukning kan visualiseres på forskellige tidspunkter under en inverteret mikroskop, helst en udstyret med et video-kamera til billede erhvervelse. Endelig, måling af ændringer i området celle-dækket af web-baseret automatiseret image analysis program giver mulighed for kvantificering af hastigheden af sår lukning og celle migration. Samlet set er denne metode et skridt fremad med hensyn til den klassiske analyse, hvor en skramme foretages manuelt af incising sammenflydende celle encellelag med en steril nål eller en pipette tip11. Ja, den sidste procedure kan ødelægge plast bunden af fadet plade/brønd og overfladebehandling, oprettelse af rynker. Derudover har området "sårede" ikke en veldefineret bredde langs hele længden af kløften, som dette stærkt afhænger af pres fra forskere til nålen/tip. Derudover kan forrykke cellerne danne klumper af levende og døde celler på kanten af bunden; Derudover kan spredning af levende celler i området "sårede" interferere med hastigheden af celle migration, genererer ikke-reproducerbare resultater12.
Desuden, takket være en ridse billede analyse platform, kan brugere hurtigt modtager (i minutter) kvantitative data om vandrende funktionsmåden for de markerede celler uden at erhverve yderligere software. Denne platform er i stand til at analysere fase kontrast mikroskopi billeder af lav (~ 5 X), medium (~ 10 X) og høj (~ 20 X) forstørrelse. Efter at uploade en zip-fil af billeder (i *.jpg, *.jpeg, *.jp2, *.png, *.gif, *.tiff format) udføres analysen automatisk for at generere en sammenfattende fil, der viser procentdelen af både celle-dækkede områder og scratch samt hastigheden af celle migration, med forskellige tidsintervaller.
I dette arbejde, ved hjælp af en frog-hud AMP-derivat, dvs Esc(1-21) og sine diastereomer Esc(1-21) - 1 c13og en bronchiale cellelinie udtrykker det funktionelle CF transmembrane ledningsevne regulator (CFTR)14,15, et eksempel på peptid-induceret celle migration sammenlignet med ubehandlet (kontrolprøverne) er fastsat. Bemærk, at luftvejene epitel og CFTR spille en afgørende rolle i at opretholde lungefunktion og sår reparation16. Derudover ved selektiv hæmmere (fx AG1478)17 af epidermal vækstfaktor receptor (EGFR), beviser, at migration af bronkial celler induceret af de førnævnte peptider indebærer aktivering af EGFR12, 18 er rapporteret.
I Resumé er målet med denne procedure at vise, hvordan brugen af sådanne silikone kultur skær repræsenterer en hurtig og lettilgængeligt assay til præcist bestemme migrationen af vedhængende celler (fx bronchiale epitelceller) og den molekylære mekanismer kontrollere sådanne begivenheder.

<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

Formålet med dette arbejde er at vise en ny metode til at vurdere muligheden af nogle immunmodulerende molekyler, såsom antimikrobielle peptider (AMP'er), at stimulere celle migration. Vigtigere, er celle migration en trafikhastighedsbegrænsning begivenhed i løbet af sårheling processen at genskabe integriteten og normal funktion af væv lag efter skade. Fordelen ved denne metode over klassisk analysen, som er baseret på en manuelt lavet bunden i en celle éncellelag, er brugen af specielle silikone kultur indsætter giver to rum for at oprette en celle-fri pseudo såret felt med en veldefineret bredde (500 μm ). Desuden, på grund af en automatiseret billede analyse platform er det muligt at hurtigt få kvantitative data om hastighed sår lukning og celle migration. Mere præcist, bliver effekten af to frog-hud ampere på migration af bronchiale epitel celler vist. Derudover vil forbehandling af disse celler med specifikke hæmmere give oplysninger om de molekylære mekanismer bag sådanne begivenheder.

Denne protokol blev brugt til at bestemme sårheling effekten af Esc(1-21) og Esc(1-21) - 1 c i celle migration aktivitet induceret på bronchiale epitel celler, der udtrykker den funktionelle CFTR. I denne analyse, kultur skær blev placeret i wells af en 12-godt plade, og hvert rum var seedet med 35.000 celler i MEMg suppleret med 10% FBS. Cellerne nåede komplet sammenløbet inden for 24 h. bagefter, et 500 μm hul blev genereret, og hver godt var fyldt med MEMg som indeholder peptid i...

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

Vi præsenterer her, en protokol for at vurdere effekten af peptider for migration af bronchiale epitel celler. Denne metode giver mulighed for hurtig og meget reproducerbare opnåelse af kvantitative data om hastighed i celle migration og sår lukning.

En roman In Vitro sår Healing Assay for at evaluere celle 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.

Research

JoVE Journal

Immunology and Infection

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

En kvantitativ vurdering af Cell Migration af Phagokinetic Track Motilitet Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Kvantitative<em&gt; In vitro</em&gt; Assay til måling neutrofiladhæsion til Aktiverede primære humane mikrovaskulære endotelceller under statiske forhold

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

In vitro assay at evaluere virkningen af immunoregulerende Pathways på HIV-specifikke CD4 T-celle effektorfunktion

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

In vitro Coculture Assay at vurdere Patogen induceret Neutrophil Trans-epitel Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Image-baserede Flowcytometri Teknik at vurdere ændringer i granulocyt Function<em&gt; In vitro</em

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

En CFSE-baserede Assay til at studere den Migration af murine Skin dendritiske celler i drænende lymfeknuder Under Infektion med<em&gt; Mycobacterium bovis</em&gt; Bacille Calmette-Guérin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Udvikling og Identifikation af en roman underpopulation af human neutrofil-afledte Giant fagocytter<em&gt; In vitro</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Anvendelse af en monocyt Monolayer Assay til Evaluer Fcy receptor-medieret fagocytose

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

En High-throughput Kompatibel Assay at evaluere Drug Effekt mod makrofag passeres<em&gt; Mycobacterium tuberculosis</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

En roman Feeder-fri ordning for masseproduktion af Murine naturlige dræberceller In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...