We want to give thanks to older members of the lab that help to improve and refine these techniques to its actual state: Dr. Celia Martinez-Mora; Dr. Anna Mrowiec, Dr. Catalina Ruiz-Ca&#241;ada and Dr. Antonia Alcaraz-Garc&#237;a. We are indebted to the Hospital Cl&#237;nico Universitario Virgen de la Arrixaca for strongly supporting the development of these techniques. Also the Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias. Plan Estatal I+D+I and Instituto de Salud Carlos III-Subdirecci&#243;n General de Evaluaci&#243;n y Fomento de la Investigaci&#243;n (Grant no.: PI13/00794); www.isciii.es. Fondos FEDER &#8220;Una manera de hacer Europa&#8221;. We also thank Universidad de Murcia, IMIB-Arrixaca and FFIS for administrative support and assistance. Finally, we want to give a special thanks to Dr. Isabel Mart&#237;nez-Argudo and the Facultad de Ciencias Ambientales y Bioqu&#237;mica, Campus Tecnol&#243;gico de la F&#225;brica de Armas, Universidad Castilla la Mancha, Toledo for their kind support in willingly ceding the Biomedicine and Biotechnology Laboratory to make possible the filmed part of this paper.

1. Artificial Wound Scratch Assay for Quantitative Studies
<ol>
	<li>Cell monolayer preparation
	<ol>
		<li>Working under sterile conditions, seed and grow Mv1Lu or HaCaT epithelial cells in culture flasks using serum-supplemented medium. Refresh medium once every 24-48 h. After cells reach 80% confluence, detach cells using an appropriate method, i.e., trypsinization9. 
		NOTE: Mv1Lu and HaCaT epithelial cell lines are cultured using EMEM and DEMEM culture medium,...

<ol>
	<li>Singer, A. J., Clark, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutaneous+wound+healing.">Cutaneous wound healing.</a> N Engl J Med. 341 (10), 738-746 (1999).</li><li>Davidson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+wound+repair.">Animal models for wound repair.</a> Arch Dermatol Res. 290, S1-S11 (1998).</li><li>Alcaraz, 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=Amniotic+Membrane+Modifies+the+Genetic+Program+Induced+by+TGFss,+Stimulating+Keratinocyte+Proliferation+and+Migration+in+Chronic+Wounds.">Amniotic Membrane Modifies the Genetic Program Induced by TGFss, Stimulating Keratinocyte Proliferation and Migration in Chronic Wounds.</a> PLoS One. 10 (8), e0135324 (2015).</li><li>Ruiz-Canada, C., 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=Amniotic+membrane+stimulates+cell+migration+by+modulating+Transforming+Growth+Factor-beta+signaling.">Amniotic membrane stimulates cell migration by modulating Transforming Growth Factor-beta signaling.</a> J Tissue Eng Regen Med. , (2017).</li><li>Schmierer, B., Hill, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGFbeta-SMAD+signal+transduction:+molecular+specificity+and+functional+flexibility.">TGFbeta-SMAD signal transduction: molecular specificity and functional flexibility.</a> Nat Rev Mol Cell Biol. 8 (12), 970-982 (2007).</li><li>Hu, Y. L., 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=FAK+and+paxillin+dynamics+at+focal+adhesions+in+the+protrusions+of+migrating+cells.">FAK and paxillin dynamics at focal adhesions in the protrusions of migrating cells.</a> Sci Rep. 4, 6024 (2014).</li><li>Martinez-Mora, C., 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=Fibroin+and+sericin+from+Bombyx+mori+silk+stimulate+cell+migration+through+upregulation+and+phosphorylation+of+c-Jun.">Fibroin and sericin from Bombyx mori silk stimulate cell migration through upregulation and phosphorylation of c-Jun.</a> PloS one. 7 (7), e42271 (2012).</li><li>Bernabe-Garcia, 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=Oleanolic+acid+induces+migration+in+Mv1Lu+and+MDA-MB-231+epithelial+cells+involving+EGF+receptor+and+MAP+kinases+activation.">Oleanolic acid induces migration in Mv1Lu and MDA-MB-231 epithelial cells involving EGF receptor and MAP kinases activation.</a> PLoS One. 12 (2), e0172574 (2017).</li><li>Huang, H. L., 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=Trypsin-induced+proteome+alteration+during+cell+subculture+in+mammalian+cells.">Trypsin-induced proteome alteration during cell subculture in mammalian cells.</a> J Biomed Sci. 17, 36 (2010).</li><li>Liberio, M. S., Sadowski, M. C., Soekmadji, C., Davis, R. A., Nelson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+tissue+culture+coating+substrates+on+prostate+cancer+cell+adherence,+morphology+and+behavior.">Differential effects of tissue culture coating substrates on prostate cancer cell adherence, morphology and behavior.</a> PLoS One. 9 (11), e112122 (2014).</li><li>Pierreux, C. E., Nicolas, F. J., Hill, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor+beta-independent+shuttling+of+Smad4+between+the+cytoplasm+and+nucleus.">Transforming growth factor beta-independent shuttling of Smad4 between the cytoplasm and nucleus.</a> Mol Cell Biol. 20 (23), 9041-9054 (2000).</li><li>Barrientos, S., Stojadinovic, O., Golinko, M. S., Brem, H., Tomic-Canic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+factors+and+cytokines+in+wound+healing.">Growth factors and cytokines in wound healing.</a> Wound repair and regeneration: official publication of the Wound Healing Society [and] the European Tissue Repair Society. 16 (5), 585-601 (2008).</li><li>Ansell, D. M., Holden, K. A., Hardman, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+wound+repair:+Are+they+cutting+it?.">Animal models of wound repair: Are they cutting it?.</a> Exp Dermatol. 21 (8), 581-585 (2012).</li><li>Gurtner, G. C., Werner, S., Barrandon, Y., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+repair+and+regeneration.">Wound repair and regeneration.</a> Nature. 453 (7193), 314-321 (2008).</li><li>Insausti, C. L., 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=Amniotic+membrane+induces+epithelialization+in+massive+posttraumatic+wounds.">Amniotic membrane induces epithelialization in massive posttraumatic wounds.</a> Wound Repair Regen. 18 (4), 368-377 (2010).</li><li>Wu, 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=Cell+cycle+arrest+in+G0/G1+phase+by+contact+inhibition+and+TGF-beta+1+in+mink+Mv1Lu+lung+epithelial+cells.">Cell cycle arrest in G0/G1 phase by contact inhibition and TGF-beta 1 in mink Mv1Lu lung epithelial cells.</a> Am J Physiol. 270 (5 Pt 1), L879-L888 (1996).</li><li>Boukamp, P., 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=Normal+keratinization+in+a+spontaneously+immortalized+aneuploid+human+keratinocyte+cell+line.">Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.</a> J Cell Biol. 106 (3), 761-771 (1988).</li><li>Marshall, J., Wells, C. M., Parsons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Migration: Developmental Methods and Protocols. , 97-110 (2011).</li><li>Nyegaard, S., Christensen, B., Rasmussen, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+method+for+accurate+quantification+of+cell+migration+using+human+small+intestine+cells.">An optimized method for accurate quantification of cell migration using human small intestine cells.</a> Metabolic Engineering Communications. 3, 76-83 (2016).</li><li>Szybalski, W., Iyer, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosslinking+of+DNA+by+Enzymatically+or+Chemically+Activated+Mitomycins+and+Porfiromycins,+Bifunctionally+"Alkylating"+Antibiotics.">Crosslinking of DNA by Enzymatically or Chemically Activated Mitomycins and Porfiromycins, Bifunctionally "Alkylating" Antibiotics.</a> Fed Proc. 23, 946-957 (1964).</li><li>Tomasz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitomycin+C:+small,+fast+and+deadly+(but+very+selective).">Mitomycin C: small, fast and deadly (but very selective).</a> Chem Biol. 2 (9), 575-579 (1995).</li><li>Sherry, D. M., Parks, E. E., Bullen, E. C., Updike, D. L., Howard, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+using+silicone+elastomer+masks+for+quantitative+analysis+of+cell+migration+without+cellular+damage+or+substrate+disruption.">A simple method for using silicone elastomer masks for quantitative analysis of cell migration without cellular damage or substrate disruption.</a> Cell Adh Migr. 7 (6), 469-475 (2013).</li><li>Dennis, E. A., Rhee, S. G., Billah, M. M., Hannun, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+phospholipase+in+generating+lipid+second+messengers+in+signal+transduction.">Role of phospholipase in generating lipid second messengers in signal transduction.</a> FASEB J. 5 (7), 2068-2077 (1991).</li><li>Lampugnani, M. G. <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+into+a+wounded+area+in+vitro.">Cell migration into a wounded area in vitro.</a> Methods Mol Biol. 96, 177-182 (1999).</li></ol>

The authors declare that there is no conflict of interest.

Upon skin or mucous membrane disruption, barrier function is restored by the actions of numerous cell types, including fibroblasts or epithelial and immune cells. Conjointly, these cells undergo a complex process involving apoptosis, proliferation, differentiation, and importantly, fibroblast and epithelial cell migration, which is the ultimate mechanism responsible for restoration of the disrupted tissue and the closure of the superficial epithelial gap1,12 Thus...

Cell migration is required for wound healing, as it is responsible for the final closure of the epithelial gap and restoration of the disrupted surface1. Performing artificial wounds in animal models allows for the replication of this complex process in near physiological conditions2. However, this approach often results in costly and complicated experimental procedures, that potentially lack precision for the study of distinct processes, due to the intricate nature of the wound-healing process.
In vitro culture of epithelial cell lines provides a helpful alternative to animal models for researching the role that these cells play in wound healing and the effects of treatment on cell migratory behavior. The physiology of epithelial cells is often studied by molecular techniques using non-confluent cultures3,4,5,6; however, the disruption of the epithelium integrity is usually achieved by fine mechanical incisions. In cell culture, this implies that negligible number of cells may be exposed to the wound gap, and they represent a too small sample for molecular biology techniques. However, these lesions can be studied at the microscopic scale, taking advantage of the innate migratory properties of some epithelial cell lines, such as the Mink Lung Epithelial cell (Mv1Lu) or the spontaneously immortalized human keratinocyte (HaCaT) cell lines.
Here we described a method for microscopy that is suitable to obtain quantitative data on the migration of epithelial cells in the context of wound healing3,4,7,8. Moreover, we present additional methods that are helpful to study qualitatively molecular and morphological changes occurring on epithelial monolayers during migration. Overall, these methods provide a framework to study both the dynamics and morphological changes involved with epithelial cell behavior and response to treatments during wound healing.

<table>
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Biowest, Nuaill&#233;, France</td>
			<td>L0102-500</td>
			<td>Optional 10 % FBS supplement</td>
		</tr>
		<tr>
			<td>Eagles&#8217;s Minimum Essential Medium (EMEM)</td>
			<td>Lonza</td>
			<td>BE12 -662F</td>
			<td>Optional 10 % FBS supplement</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Lonza</td>
			<td>BE17-605E</td>
			<td>Use at 2 mM</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Thermo Fisher Scientific, Waltham, MA USA</td>
			<td>DE17603A</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>T4049</td>
			<td>Dilute as appropriate</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>P9155</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (DPBS) (10x)</td>
			<td>Gibco by Life Technologies</td>
			<td>14200-067</td>
			<td>Dilute to 1x</td>
		</tr>
		<tr>
			<td>24-well culture plates</td>
			<td>BD FALCON//SARSTED</td>
			<td>734-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plates</td>
			<td>SARSTEDT</td>
			<td>83-3920</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor (EGF)</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>E9644</td>
			<td>Used 10 ng/mL</td>
		</tr>
		<tr>
			<td>Round cover glass</td>
			<td>MENZEL-GL&#196;SER</td>
			<td>MENZCB00120RA020</td>
			<td>SHORT DEPTH OF FIELD</td>
		</tr>
		<tr>
			<td>Reinforced razor blade no. 743</td>
			<td>Martor (through VWR)</td>
			<td>MARO743.50</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;l sterile aerosol pipet tips</td>
			<td>VWR</td>
			<td>732-0541</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#181;l sterile aerosol pipet tips</td>
			<td>VWR</td>
			<td>732-0528</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital camera coupled phase contrast microscope</td>
			<td>Motic Spain</td>
			<td>Moticam camera 2300 3.0 M Pixel USB 2.0; Motic Optic AE31</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>ZEISS Microimaging, Germany</td>
			<td>LSM 510 META</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm Culture dish</td>
			<td>BD FALCON</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit polyclonal anti c-Jun antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-1694</td>
			<td>Used 1:100</td>
		</tr>
		<tr>
			<td>Anti-rabbit IgG (polyclonal goat ) AF 488</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td>Used 1:400</td>
		</tr>
		<tr>
			<td>Hoechst-33258</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>14530</td>
			<td>Used 1:1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 phalloidin (in methanol) (red)</td>
			<td>Invitrogen</td>
			<td>A12381</td>
			<td>Used 1:100</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-2323</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk</td>
			<td>BD DIFCO</td>
			<td>232100</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health, USA</td>
			<td>Release 1.50i</td>
			<td></td>
		</tr>
		<tr>
			<td>Zen LSM 510 image processing software</td>
			<td>ZEISS Microimaging, Germany</td>
			<td>Release 5.0 SP 1.1</td>
			<td></td>
		</tr>
	</tbody>
</table>

microscopy based methods for the assessment of epithelial cell migration during in vitro wound healing

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

Artificial Wound Scratch Assay for Quantitative Studies: Assessing Epidermal Growth Factor (EGF) Promotion of Migration:
EGF is a well-known inducer of epithelial cells proliferation and migration, and thus a positive control for quantifying migration promotion. Mv1Lu and HaCaT cell monolayers were used in the wound scratch assays and pre-treatment pictures were obtained. After inoculation with 10 ng/mL EGF, cells were incubated fo...

Watch this Scientific Journal Video about Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing at JoVE.com

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

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

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

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

The overall goal of these epithelial cell culture artificial wounding procedures is to study cell migration from both a quantitative and qualitive perspective, allowing assessment of the effects of specific treatments of interest on wound healing. This method can help answer key questions in the wound-healing field, as they allow the precise characterization of the roles of specific migration processes during epithelial disruption. The main advantage of this approach is that it allows the correlation of cell migratory measurements with changes in the behavior of relevant molecular markers.Implication of these techniques extend towards a therapy of clinical wounds, as it offers a convenient setup for early drug testing and for wound closure. Generally in to this method will start because of difficulties in obtaining consistent reproduceability in the wounds with an generation. For an artificial wound scratch assay, begin by seeding each well of a culture plate with two milliliters of epithelial cells of interest in complete medium.Enculture the cells at 37 degrees Celsius and five percent carbon dioxide for two to three days until 100%confluency. When the cells are ready, wash the cells two times with fresh serum-free medium followed by 24 hours of culture in fresh serum-free medium. The next day, place the plate in a sterile work area and drag the narrow end of a sterile micro-pipette tip across the bottom of each well two times to generate a cross-shaped wound.A consistent wound gap is achieved by applying constant, firm pressure while smoothly moving the tip along the epithelial surface. Excessive pressure will deform the tip, causing an irregular wound radius. Gently remove the supernatant to discard the detached cells and carefully add fresh serum-free medium to the wells.Using in inverted phase contrast microscope connected to a charge coupled device camera, align the edge of the microscope field with the adjacent intersection of the scratches to obtain up to four pre-treatment reference images of the scratch gap in each well. When all of the images have been acquired, replace the medium in the designated treatment wells with fresh medium, supplemented with selected treatments of interest and return the plates to the cell culture incubator. When at least one condition reaches about 90%scratch gap closure, gently replace the supernatant with one milliliter of four percent formalin in PBS for a 15 minute incubation at room temperature.Then gently wash the cells with PBS at least three times to remove any excess formalin and image the wells as just demonstrated, using the same imaging parameters in the original reference areas captured after scratching. To analyze the images using the appropriate image processing software, open the recorded, pre-treatment image of interest and under the Image menu, set the image type to 8-bit. Under the Process menu, select the Filters sub-menu and apply Variance filtering.Under the Image menu, open the Adjust sub-menu and set the Threshold to black and white. In the Process menu, select the Binary sub-menu and apply Fill Holes. Under the Analyze menu, select Set Measurements and activate Area.Then draw the measurable area, following the migrating edge contour to delimit the gap. Under the Analyze menu, select Analyze Particles and record the total area values for the drawn area surface. To quantify the absolute migration for each set of individual samples as the difference between the gap surface measurements, export the pre and post-treatment total gap surface area values to a spreadsheet and plot the quantification outputs.For an artificial migration front assay, under sterile conditions, add one layer of up to 33 sterilized round coverslips into empty 10 centimeter culture plates. When the plate surface is completely covered, gently seed the epithelial cells of interest at a concentration that allows for 100%confluence after two or three days of culture. If necessary, gently depress the coversips with a sterile micropipette tip to prevent floating.When the cells reach confluency, replace the supernatant with serum-free medium for another 24 hours of culture. Then, use sterile tweesers to carefully transfer one coverslip into a new 10 centimeter plate containing fresh serum-free medium, taking care to hold the coverslip along its periphery. To create the artificial wounds, drag a sterilized razor blade back and forth across each coverslip to make a three to four millimeter transverse line over the center of each cell culture to completely remove the central monolayer strip.Take care to place the razor blade edge heavily across the coverslip surface when making the wound to safeguard against an irregular edge shape and the generation of flopping epithelial portions. Transfer up to four wound coverslips, cell side up, into individual wells of a six well plate, containing at least two milliliters of serum-free medium and gently wash each well two times with fresh serum-free medium to remove any detached cells. When all of the detached cells have been discarded, gently replace the wash medium with fresh medium supplemented with the treatments of interest, and place the plate in the cell culture incubator.At the end of the appropriate experimental period, fix the cells with four percent formalin in PBS as just demonstrated, and stain the cells with the appropriate fluorescent conjugated antibodies of interest. Then image the structural changes occurring at the migrating front edge by fluorescence microscopy according to the experimental parameters of interest. In this experiment, the wound gaps were measured before and after treatment with epidermal growth factor, a well-known inducer of epithelial cell proliferation and migration.The absolute migration was then calculated as the difference between the pre and post-treatment gap surface areas. Treatment with epidermal growth factor potentiates cell cytoskeletal dynamics as observed in these laser scanning microscopy images of F-Actin staining of control and growth factor stimulated cells. Further, c-Jun over-expression is observed with an increased expression of the protein apparent in the cells adjacent to the migrating front.While attempting this procedure, it's important to remember to check the integrity of the wound edges for the presence of epithelial cell flaps that may disrupt the migration quantification. After watching this video, you should have a good understanding of how to both quantitatively and qualitatively assess the migratory behavior of the

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9.1K visualizações.  IMIB-Arrixaca. The overall goal of these epithelial cell culture artificial wounding procedures is to study cell migration from both a quantitative and qualitive perspective, allowing assessment of the effects of specific treatments of interest on wound healing. This method can help answer key questions in the wound-healing field, as they allow the precise characterization of the roles of specific migration processes during epithelial disruption. The main advantage of this approach is that it allows the cor...