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

Detta arbete stöds av finansiering från Sapienza University of Rome och italienska cystisk fibros Research Foundation (projektet FFC nr 11/2014 antogs av FFC delegationer från Siena, Sondrio Valchiavenna, Cerea Il Sorriso di Jenny och Pavia). En del av detta arbete fick också stöd av FILAS Grant Prot. FILAS-RU-2014-1020.
Vi är tacksamma att Dr Loretta Ferrera (U.O.C. Genetica Medica, Istituto Giannina Gaslini, Genova, Italien) för att tillhandahålla bronkial epitelceller.

1. cell förberedelse
<ol><li>Frö 2.5x106 celler i 10 mL av minst viktigt Medium (MEM) kompletteras med 2 mM glutamin (MEMg), plus 10% fetalt bovint serum (FBS), antibiotika (0,1 mg/mL av penicillin och streptomycin) och puromycin (0,5 µg/mL för urval och underhåll av den cellinje) i en T75 kolv. Inkubera kolven vid 37 ° C och 5% CO2 i 2 dagar. Innan du börjar experimentet, kontrollera sammanflödet av celler under ett inverterat Mikroskop. Obs: De celler som används för experiment är ...

<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>

Cellmigration är en viktig process i många fysiologiska och patologiska händelser inklusive sårläkning, embryonal utveckling och cancer metastaser. Den grundläggande proceduren att studera cell migration i vitro innebär: (i) skapandet av en cell enskiktslager, (ii) produktionen av en pseudo såret i konfluenta lagret av celler, (iii) att fånga bilder vid olika tidpunkter tills såret stängning nås , och (iv) analys av den bildsekvens för att kvantifiera flyttning hastigheten av valda celler.
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Det är i hög grad känt att sårläkning hos djur är en grundläggande process att återupprätta integritet och normal funktion av vävnad skikt efter skada1. Trots epitelial ytor som utsätts för den yttre miljön (t.ex. huden, andningsorganen, och mage och tarm) bildar en skyddande barriär från fysiska och kemiska förolämpningar, bildandet av sår kan lätt uppstå, särskilt efter kirurgi eller mikrobiella infektioner2. Som ett exempel leder koloniseringen av lungvävnad av opportunistiska bakterie patogenen Pseudomonas aeruginosa, särskilt i lider av cystisk fibros (CF), till skador i luftvägarna epitel med åtföljande andningssvikt3, 4. Sårläkning är en komplex värd reparation mekanism att återställa en skadad vävnad5normala arkitektur. Det kännetecknas av inledande inflammation, följt av en regenerering bemyndigades epithelialization, angiogenes och vävnad remodeling med produktionen av kollagen och cell differentiering6,7,8 . Att säkerställa epitelial integritet och för att kontrollera mikrobiell spridning, producera alla levande organismer försvar molekyler, inklusive antimikrobiella peptider (ampere)9,10. Sårläkningsprocessen är mycket svårt att simulera in vitro- på grund av cellfragment och komplexa samspel mellan olika celltyper. Dock i vitro förmåga en peptid att påskynda nedläggningen av ett pseudo sår genom att stimulera migration av epitelceller är ett tecken på dess förmåga att läka en komprometterad epitel. Faktiskt, cellmigration är en hastighetsbegränsning händelse i sårläkning och studerar faktorer som kan påverka cellmigration kommer att bidra till målet terapier för förbättrad sårläkning.
Här, tillhandahålls en mycket reproducerbara experimentell analys baserat på speciella silikon kultur skär att utvärdera cell migration in vitro. Den bygger på skapandet av en 500 μm gap (pseudo såret) på en konfluenta cell enskiktslager. Cellerna i kanten av fältet konstgjorda ”sårade” börjar migrera till området cellfria, bildar nya cell cell kontakter. Kultur skäret representerar ett nytt verktyg för snabb sårläkning experiment. Två reservoarer åtskilda av en 500 μm vägg tillhandahålls, och de kan placeras korrekt i en 3 cm maträtt tallrik eller i brunnen av en 12-bra platta. Fylla varje fack insatsen med en cellsuspension tillåter celler att växa i varje utsedda området tills confluence, medan avlägsnandet av skäret kommer att skapa en ren cellfria lucka på cirka 500 μm (samma bredd som den avskiljande väggen). En ordentlig cellodlingsmedium kompletteras med en test-förening kan sedan läggas in i skålen plattan per brunn. Efteråt, gap nedläggningen kan visualiseras vid olika tidpunkter under en inverterade Mikroskop, helst en utrustad med en videokamera för bild förvärv. Slutligen, mätning av förändringar i området cell-omfattas av programmet web-baserad automatiserad bild analys kan kvantifiering av hastigheten på såret stängning och cell migration. Sammantaget är denna metod ett steg framåt när det gäller klassiska analysen, där en repa görs manuellt av öppning konfluenta cell enskiktslager med en steril nål eller en pipett tips11. Faktiskt den sista proceduren kan förstöra plast botten av skålen plattan/brunn och ytbeläggningen, skapa rynkor. Dessutom har ”sårade” området inte en väldefinierad bredd längs hela längden av klyftan, eftersom det beror mycket på trycket tillämpas av forskare på nålspetsen /. Dessutom kan rubbas cellerna bilda klumpar av levande och döda celler på kanterna av scratch; Dessutom kan spridningen av levande celler i området ”sårade” påverka hastigheten av cellmigration, genererar icke-reproducerbara resultat12.
Dessutom, tack vare en scratch bild analysplattform, kan användare snabbt få (inom minuter) kvantitativa uppgifter om flyttande beteendet för de markerade cellerna utan nödvändigheten av att förvärva ytterligare programvara. Denna plattform är kapabel att analysera fas kontrast mikroskopi bilder av låg (~ 5 X), medium (~ 10 X) och hög (~ 20 X) förstoring. Efter uppladdning en zip-fil av bilder (i *.jpg, *.jpeg, *.jp2, *.png, *.gif, *.tiff format) sker analysen automatiskt för att generera en summary fil som visar procentandelen av både cell-täckt och scratch områden, liksom hastigheten på cell migration, vid olika tidsintervaller.
I detta arbete, med hjälp av en groda-hud AMP-derivat, dvs Esc(1-21) och dess diastereomer Esc(1-21) - 1 c13och en bronkial cellinje som uttrycker den funktionella CF transmembrane conductance regulator (CFTR)14,15, ett exempel på peptid-inducerad cellmigration jämfört med obehandlade (kontrollproven) tillhandahålls. Observera att de luftvägarna epitel och CFTR spela en avgörande roll för att upprätthålla lungfunktion och såret reparation16. Dessutom genom selektiv hämmare (t.ex. AG1478)17 av epidermal tillväxtfaktor receptor (EGFR), bevis att migreringen av bronkial celler induceras av ovannämnda peptiderna innebär aktivering av EGFR12, 18 redovisas.
Sammanfattningsvis är målet med detta förfarande att visa hur användningen av sådana silikon kultur skär representerar en snabb och lättillgänglig analysen att exakt fastställa migration av vidhäftande celler (t.ex., bronkial epitelceller) och molekylära mekanismer för kontroll av sådana händelser.

<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

Syftet med detta arbete är att visa en ny metod för att utvärdera förmågan hos vissa immunmodulerande molekyler, såsom antimikrobiella peptider (ampere), att stimulera cellmigration. Viktigt, är cellmigration en hastighetsbegränsning händelse under sårläkning processen att återupprätta integritet och normal funktion av vävnad skikt efter skada. Fördelen med denna metod jämfört med klassisk analysen, som är baserad på en manuellt gjorda repa i en cell enskiktslager, är användningen av speciella silikon kultur skär som ger två fack för att skapa ett cellfria pseudo sår fält med en väldefinierad bredd (500 μm ). På grund av en automatiserad bild analysplattform är det dessutom möjligt att snabbt få kvantitativa uppgifter om hastigheten på såret stängning och cell migration. Mer exakt, visas effekten av två groda-hud ampere på migration av bronkial epitelceller. Dessutom ger förbehandling av dessa celler med specifika hämmare information om de molekylära mekanismerna bakom sådana händelser.

Detta protokoll användes för att bestämma de sårläkning effekt av Esc(1-21) och Esc(1-21) - 1 c när det gäller cell migration aktivitet inducerad på bronkiell epitelceller som uttrycker den funktionella CFTR. I denna analys, kultur skär placerades i brunnar 12-well platta, och varje fack var seedad med 35.000 celler i MEMg kompletteras med 10% FBS. Cellerna nått fullständig sammanflödet inom 24 h. efteråt, en 500 μm lucka genererades och var väl var fylld med MEMg som inneh...

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

Här presenterar vi ett protokoll för att utvärdera effekten av peptider för migration av bronkial epitelceller. Denna metod möjliggör snabba och mycket reproducerbara obtainmenten av kvantitativa uppgifter om hastigheten på cell migration och såret stängning.

En roman In Vitro Wound Healing analys för att utvärdera cellmigration

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 utvärdering av cellmigration genom Phagokinetic Track Motility analys

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

Kvantitativ<em&gt; In vitro</em&gt; Analys för att mäta neutrofilvidhäftning till Aktiverade primära humana mikrovaskulära endotelceller under statiska förhållanden

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-analys för att utvärdera effekten av immunPathWays på HIV-specifika CD4 T-cell 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 Kokulturanalys för att bedöma patogeninducerad neutrofil transeptelmigration

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

Bild-baserade flödescytometri Technique att utvärdera förändringar i Granulocyt Funktion<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-baserad analys för att studera migration av dendritiska Murin hudceller i dränerande lymfkörtlar under infektion med<em&gt; Mycobacterium bovis</em&gt; Bacille Calmette-Guerin

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

Utveckling och identifieringen av en ny underpopulation av humant neutrofilt härrörande Giant fagocyter<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

Användning av en monocyt Monolayer analys för att utvärdera Fcy-receptor-medierad fagocytos

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 hög kapacitet Kompatibel analys för att utvärdera läkemedlets effektivitet mot makrofag passe<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-fria System för massproduktion av murina naturliga mördarceller 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 ...