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, respectively, supplemented with 2 mM L-Glutamine, and incubated at 37 &#176;C with a controlled atmosphere of 5% CO2. Every cell line must be assayed thoroughly to determine cell concentration and timing required to reach full confluency. Typically for Mv1Lu or HaCaT cells, 2-4 x 104 or 4-6 x 104 cells/well, respectively, are seeded in a 175 cm2 culture flask, and at 80% confluence yields up to 35 x 106 Mv1Lu or 1 x 107 HaCaT cells.</li>
		<li>In either a 12-well or 24-well culture plate, seed in each well 2 mL of cells. Refresh medium once every 24-48 h. Ensure cell numbers are high enough to reach 100% confluence after 2 or 3 days.</li>
		<li>After the cells reach confluence, remove the serum-supplemented medium and wash twice with fresh serum-free medium. Keep cells in serum-free medium for 24 h before the scratching. 
		NOTE: Mv1Lu or HaCaT cells do not have special substrate requirements; however, for cell lines susceptible to detaching spontaneously in serum-free conditions, pre-coating of the culture plate surface using a poly-L-lysine solution should be considered10.</li>
	</ol>
	</li>
	<li>Monolayer scratching
	<ol>
		<li>Working in a sterile environment, scratch the culture monolayer by firmly dragging the narrow end of a 20 &#181;L or 200 &#181;L sterile micropipette tip, depending on the desired scratch width. Keep the tip perpendicular to the culture surface to maximize gap homogeneity.
		<ol>
			<li>Place the culture plates over a dark surface to clearly monitor the cell monolayer. If needed, perform two perpendicular scratches (cross-shaped) on each well to study up to 4 migration areas.</li>
		</ol>
		</li>
		<li>Wash detached cells by gently removing culture medium and gently adding fresh serum-free medium. Repeat two times, or until most unattached cells have been removed.</li>
	</ol>
	</li>
	<li>Experimental procedure and data collection
	<ol>
		<li>Take up to 4 pre-treatment reference images centered on the scratch gap from each well using an inverted phase-contrast microscope incorporating a CCD camera. Select interest areas by aligning the edge of microscope field to the adjacent intersection of scratches. 
		NOTE: Typically, images are acquired using a 10x magnification and a 1,280 x 1,024 pixel image size. Selecting interest areas as described above helps for easy localization and validation of up to 4 measurements per well.</li>
		<li>Once all the images are acquired, designate treatment wells; exchange the medium in the wells with fresh medium that contains selected treatments. 
		NOTE: Alternatively, for chemicals or compounds that do not disturb culture medium homogeneity, treatments can be inoculated directly into the culture medium.</li>
		<li>Incubate the scratched plate (37 &#176;C with a controlled atmosphere containing 5% CO2) until the conditions under observation reach 90% scratch gap closure. Avoid full closure. 
		NOTE: Total gap closure nullifies post-treatment picture collection as reference areas are not detectable and the time reference for gap closing cannot be established. In the case of Mv1Lu or HaCaT cells, 16-19 h are required to reach 90% confluence.</li>
		<li>Stop cell migration by fixing the cells; gently replace the experimental culture medium with 1 mL of 4% formalin in PBS. Incubate for 15 min at RT.</li>
		<li>Wash the cells to remove excess formalin; gently replace the solution in the wells with fresh PBS. Repeat at least two times. 
		NOTE: At this stage, after sealing, plates can be stored at 4 &#176;C indefinitely.</li>
		<li>Take post-treatment reference images of each well from the original reference areas captured after scratching. Use the same equipment (inverted optical phase-contrast microscope incorporating a CCD camera) and matching picture settings (magnification and digital resolution/density). 
		NOTE: For cells that migrate individually and fill the gap independently of the formation of a coherent front (e.g., MDA-MB-231 breast cancer human cells), the time-course images may allow the calculation of individual cell migration speeds.</li>
	</ol>
	</li>
	<li>Image analysis and quantification of migration
	<ol>
		<li>Using image processing software (e.g., ImageJ), define scratch gap limits and determine the gap surface for up to four measurements in pre-treatment pictures. Record the data as &#34;pre-treatment gap surface&#34; (PREGAP). Repeat the same procedure for post-treatment pictures. Record the data as &#34;post-treatment gap surface&#34; (POSTGAP).
		<ol>
			<li>Open the recorded image using ImageJ. In the &#34;image&#34; menu, set image type to 8 bit. In the &#34;process&#34; menu, go to &#34;filters&#34; submenu and apply &#34;variance&#34; filtering.</li>
			<li>In the &#34;image&#34; menu, enter &#34;adjust&#34; submenu and set threshold to black and white (B&#38;W), make sure &#34;Dark background&#34; is not selected. In the &#34;process&#34; menu, go to &#34;binary&#34; submenu,&#160; and select &#34;fill holes&#34;.</li>
			<li>In the &#34;analyze&#34; menu, select &#34;set measurements&#34; and activate &#34;area&#34;. Then draw the measurable area following the migrating edge contour thus delimiting the gap.</li>
			<li>In the &#34;analyze&#34; menu, select &#34;analyze particles&#34; and record &#34;total area&#34; values for the drawn area surface.</li>
		</ol>
		</li>
		<li>Introduce PREGAP and POSTGAP total area values in a spreadsheet to quantify absolute migration for each set of individual samples as the difference of gap surface measurements: PREGAP &#8722; POSTGAP [Arbitrary units]. Additionally, normalize absolute migration data of each condition to control samples: (SAMPLE / CONTROL) * 100 [%]. 
		NOTE: For cells that migrate individually and fill the gap independently of the formation of a coherent front, (e.g., MDA-MB-231 breast cancer human cells), the absolute migration can be calculated by counting cells invading a central strip either in the control or the treated samples.</li>
		<li>Plot quantification outputs (see Figure 1). Perform statistical analysis if necessary. Consider Student&#39;s t-test when comparing two conditions or Analysis of Variance test for greater number of conditions.</li>
	</ol>
	</li>
</ol>
2. Artificial Migration Front Assay for Topographical Studies
<ol>
	<li>Cell monolayer preparation
	<ol>
		<li>Working in sterile conditions, place one layer of sterilized round coverslips in empty culture plates until the plate&#39;s surface is completely covered. 
		NOTE: Up to 12 and 33 coverslips fit on 5 cm and 10 cm diameter plates, respectively.</li>
		<li>On the culture plate, gently seed an adequate volume of cells at a concentration that allows 100% confluence after 2 or 3 days. Make sure no coverslip overlaps neighboring coverslips and prevent coverslips from floating by applying gentle pressure with a sterile micropipette tip. Refresh the medium every 24-48 h. 
		NOTE: Every cell line must be assayed thoroughly to determine cell concentration and timing required to reach full confluency. Typically for Mv1Lu or HaCaT cells, 2-3 x 106 or 2.5-4 x 106 cells/10 cm-diameter plate, respectively, are seeded. For smaller plates, cell numbers must be scaled down.</li>
		<li>After the cells reach confluence, remove the serum-supplemented medium and replace with fresh serum-free medium. Keep the cells in serum-free medium for 24 h before the artificial wound is performed. 
		NOTE: Mv1Lu or HaCaT cells do not have special substrate requirements; however, for cell lines susceptible of detaching spontaneously in serum-free conditions, pre-coating of the culture surface using a poly-L-lysine solution should be considered10.</li>
	</ol>
	</li>
	<li>Monolayer wounding
	<ol>
		<li>Using sterile tweezers, gently move a coverslip to a clean 10 cm plate containing fresh serum-free medium. Avoid damaging the monolayer by holding the coverslip at the periphery with tweezers. Avoid excessive pressure that could result in coverslip breakage.</li>
		<li>Create artificial wounds by dragging a sterilized razor blade in a transverse line over the center of the coverslips. Drag 3-4 mm back-and-forth, in order to completely remove the central monolayer strip. Make sure no cell debris remains attached to the wounded edges. 
		NOTE: On a 12-mm diameter round coverslip, the incision is made at the mid of the coverslip. Make sure to leave a streak of cells opposite the main front large enough (at least 2 mm wide) to avoid tilting of the coverslip in the following steps.</li>
		<li>Transfer wounded coverslips with cells facing up, to a clean 6-well plate containing at least 2 mL fresh serum-free medium. Fit up to 4 wounded coverslips/well.</li>
		<li>Gently wash two times using fresh serum-free medium to remove detached cells.</li>
	</ol>
	</li>
	<li>Experimental procedure and sampling
	<ol>
		<li>Gently exchange the medium in the wells with fresh medium that contains selected treatments. 
		NOTE: Alternatively, for chemicals or compounds that do not disturb culture medium homogeneity, treatments can be inoculated directly into the culture medium. Proceed with caution to avoid any potential cell detachment.</li>
		<li>Keep the plate inside a cell incubator (37 &#176;C, 5% CO2) for the desired experimental timing.</li>
		<li>Stop cell migration by fixing the cells; gently replace the experimental culture medium with 1 mL of 4% formalin in PBS. Incubate for 15 min at RT. 
		NOTE: For time course experiments, remove the samples from the culture plate and fix them in a separate plate to avoid formalin fumes affecting the other, ongoing experimental conditions.</li>
		<li>Wash the cells to remove excess formalin; gently replace the solution in the wells with fresh PBS. Repeat at least two times. 
		NOTE: At this stage, after sealing, plates can be stored at 4 &#176;C indefinitely.</li>
	</ol>
	</li>
	<li>Immunofluorescence (IF) and topological analysis
	<ol>
		<li>Perform IF staining and imaging on individual coverslips as described elsewhere3,4,11.
		<ol>
			<li>Permeabilize for 10 min by immersing coverslips in a 0.3% Triton X-100 solution in PBS.</li>
			<li>Block for 30 min using the following blocking solution: 0.3% Bovine Serum Albumin; 10% Fetal Bovine Serum; 5% skim milk; 0.3% Triton X-100 solution in PBS. 
			NOTE: Blocking solution without milk can be prepared in advance and stored at -20 &#176;C.</li>
			<li>Incubate for 1 h at room temperature inside a moist chamber, with primary antibody diluted in milk-free blocking solution. Place the coverslips upside-down in the 15 &#181;L antibody solution to ensure proper distribution. 
			NOTE: The antibody working dilution is variable and will depend on the antibody in use. For example, the Rabbit anti-c-Jun antibody requires a 1/100 dilution.</li>
			<li>Wash three times by dipping the coverslips in a in 0.1% Triton X-100 solution in PBS.</li>
			<li>Incubate the coverslips in secondary antibody diluted in milk-free blocking buffer for 30 min. 
			NOTE: The antibody working dilution is variable and will depend on the antibody in use. For example, the Goat-anti-Rabbit (488) requires a 1/400 dilution for optimal resolution. Other labeling reagents such as phalloidin and Hoechst-33258 can be added to the incubation buffer at this stage.</li>
			<li>Wash three times by dipping the coverslips in 0.1% Triton X-100 solution in PBS.</li>
			<li>Mount the coverslips upside-down in a 10 &#181;L mounting media drop (e.g., Vectashield) placed on a microscope slide. Leave the slides in the dark at room temperature overnight for the mounting medium hardening. Afterwards, store at 4 &#176;C in the dark indefinitely.</li>
		</ol>
		</li>
		<li>Obtain either epifluorescence or confocal microscopy images of structural changes occurring at the migrating front edge. 
		NOTE: Topological studies can be performed by tiling pictures from the migrating front to the inner monolayer. Semi-quantitative studies are possible by software based analysis on local fluorescence intensities.</li>
	</ol>
	</li>
</ol>

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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. 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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, studying cell migration helps precisely describe the physiological behavior of epithelial cells, while also determining the modulatory potential of treatments for wound healing.
Performing artificial wounds on different research animal models allows for the replication of this complex process in near physiological conditions2, and thus the assessment of some pathological conditions or the effects of pharmaceutical treatments13. However, this approach often results in costly and complicated logistics, as well as burdensome experimental procedures that may generate data in a delayed fashion13. On the contrary, in vitro cell culture provides a more convenient framework for researching the behavior of cells involved in the wound healing process. While primary cell culture of epithelial cells constitutes a viable option, it can be difficult to obtain and handle the cells, which can complicate data collection. For instance, in addition to the typical low-passage restriction of primary cultures, primary human keratinocytes need a fibroblast feeding layer for growth in vitro3. On the contrary, well-established epithelial cell culture lines, like Mv1Lu or HaCaT, constitute an obstacle-free research model that features behavior and characteristics like those in primary culture cells3,4,14,15. Precisely, for the study of wound healing, these two cell lines retain critical traits in their ability to migrate and stop proliferation due to cell-to-cell contact inhibition upon confluence16,17.
Scratch assays are a reliable and versatile method for quantifying the migratory capabilities of different cell types, especially epithelial and cancer cells. For the case of cancer cells, the scratch method has been proposed for assessment of invasive potential. However, this approach falls short as an indicator for invasiveness when compared to more specific methods relying on the expression of metalloproteases needed for penetrating the extracellular matrix18,19. On the contrary, scratch assays are reliable for assessing the migratory performance of epithelial cells and the effect of different treatments on this trait. In contrast to cancer cells lines, which frequently show poor cohesion and the ability to grow on several layers, Mv1Lu, HaCaT, and other epithelial cell lines form tightly associated &#34;growth islands&#34;, which expand their margins in a process depending on both proliferation, but more importantly on cell migration. Consequently, sparse or sub-confluent cell density conditions are conventionally used for studying the effects of different treatments on the migratory physiology; especially when subsequent molecular biology techniques are applied, as this cell environment minimizes contact inhibition while maximizing the ratio of actively migrating cells. However, for epithelial cells, non-confluent conditions pose the risk of excluding important pre-existing contact inhibition influences, for instance in the form of epigenetic regulation. Thus, to study wound healing, confluent conditions confer increased fidelity of the model.
To obtain precise estimates, concomitant factors affecting the conformation of the epithelial cell layer or contributing to migration should be addressed. While confluence is desired, it is important to avoid excessive cell density or multilayered cultures, since these conditions may affect negatively. Therefore, proliferation is commonly controlled by starvation in serum-free media or by the addition of chemicals like Mitomycin C, which irreversibly arrest cell proliferation by DNA crosslinking3,20,21. The use of one or the other relies on the behavior of specific cell lines; Mitomycin C is especially indicated when reduced serum supplementation is required to avoid massive cell detachment. That is the case for HEK-293T cells, where pre-coating the culture surface using poly-L-lysine is a good option to avoid Mytomycin C treatment. The release of inflammatory factors that could alter migration also needs to be addressed. Different strategies have been used, mainly based on silicone inserts to mimic the disruption of the epithelial surface achieved with the scratch assay22. While inserts generate pre-existing gaps that allow cell migration after removing the silicone piece, the scratch method relies directly on removing some of the epithelial monolayer to generate the gap. Preference on how to generate the gap relies on the ability of inserts to protect the culture surface coating (i.e., culture plate or poly-L-lysine) from damage during scratching, while also minimizing the release of factors from damaged cells that may compromise the cell culture behavior23. Although these characteristics might be useful for some experimental settings, i.e., assessing the ability of cancer cells to migrate, in our experience we found significant drawbacks for using inserts in wound healing studies. Mv1Lu cells are capable of attaching to the silicone insert outer surface, which results in unintentionally tearing of the cell monolayer upon removal of the silicone pieces. Also, although HaCaT cells do not attach to silicone, we found a diminished ability for these cells to migrate into insert-generated gaps. To that extent, inflammation and migration are tightly associated responses, and these appear to be critical together in the context of wound healing in HaCaT cells. Moreover, since applying a plastic tip for scratching the culture surface has no apparent consequence on the ability of cells to repopulate the gap, in the case of HaCaT cells, such behavior suggests that extracellular matrix remnants in the gap may guide migration in a similar fashion as dermis in a natural wound. However, the main source of variation remains the scratching procedure itself, as the use of a plastic tip to make the wound does not always render the same size gap. We have observed variations up to 20% among different samples, due to inconsistency of pressure and position of the tip at the time of scratch. These variations can be addressed by wounding the monolayer with a more rigid object; however, any scratch on the plastic surface would jeopardize the free movement of the cells. So, variations in the initial scratch must be taken into account to calculate migration; typically, by increasing the number of measurement per condition.
In addition to quantification, applying wounding procedures together with classic IF techniques can qualitatively assess the molecular processes of cell migration. The capability of epithelial cells to grow on coverslips is well-know; in the past, variations of the scratch assays involved cells migrating on coverslips exposed to different conditions followed by fixing and staining for comparison within the wound-scratch experimental setup24. Here, we found that creating wide gaps in monolayers growing on coverslips allows for extending the experimental time courses3,4,7. Moreover, in these experiments, IF increases the detection capabilities of the procedure to potentially study any precise cellular and subcellular mechanisms involved, and it is only limited by the availability of antibodies suitable for this technique and the cells of interest. Samples on the coverslips used for IF are mounted on microscope slides, which allow for extended preservation. This aids the application of experimental setups that require long study periods, like the &#34;tiling&#34; approaches showcased here and elsewhere3,4,7. While the &#34;tiling&#34; strategy integrates spatial patterns with temporal data for reconstructing the mechanisms involved in migration, the implementation of software analysis tools can provide semi-quantitative assessments based on local fluorescence intensity, and further enrich the quality of the information obtained. In our experience, tiling also offers the possibility of studying cell-positional information for the expression of proteins in this type of assay. This positional information has been shown to be crucial to understand and better translate the effects of different agents on cell migration, especially in setups where the position of epithelial cells is very important to determine its migratory behavior3,4,14,15.
Both procedures described demonstrate great convenience by uncomplicated implementation, as well as producing quality data. Thus, these approaches that are based on microscopy studies, offer a unique and powerful framework to study both the dynamics and morphological changes involved in epithelial cell behavior and response to treatments during wound healing.

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&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>Biowest, Nuaill&amp;eacute;, France</td><td>L0102-500</td><td>Optional 10 % FBS supplement</td></tr><tr><td>Eagles&amp;rsquo;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&#039;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&amp;Auml;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 &amp;micro;l sterile aerosol pipet tips</td><td>VWR</td><td>732-0541</td><td></td></tr><tr><td>20 &amp;micro;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 for 19 h before fixation and post-treatment pictures. Proliferation was kept to a minimum by maintaining serum starvation conditions. Both pre- and post-treatment sets of pictures were analyzed using ImageJ and the central gap areas were determined. The experiment was run in duplicate, registering four measurements for each well. Quantification of the absolute migration was calculated as the difference of surfaces: (PREGAP &#8722; POSTGAP). There was a greater migratory potential in the stimulated Mv1Lu cells (Figure 1A) compared to the stimulated HaCaT cells (Figure 1B). The differences are explained by the cell sources, being mucosa epithelial cells and skin keratinocytes, respectively.
Artificial Migration Front Assay for Topographical Studies: Variations in Cytoskeletal Conformation and Spatial Expression of C-Jun:
Cell migration involves continuous and deep changes of the cytoskeleton structure in order to sustain cell displacement. HaCaT cell monolayers were used in the migration front assays under control and stimulation conditions. Treatment with EGF potentiates the cytoskeletal dynamics, which are observed by IF and laser scanning microscopy (LSM; Figure 2A). EGF treatment also results in robust Mitogen-Activated Protein (MAP) kinases signaling and c-JUN overexpression. Tiling LSM images allows for the configuration of spatial patterns. Increased expression of c-JUN at cells adjacent to the migrating front can be easily revealed by software image analysis; here, we implemented a rainbow scale for the green channel, which correspond to c-JUN labeling (Figure 2B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56799/56799fig1.jpg" /> 
Figure 1. Promotion of migration by EGF in Mv1Lu and HaCaT cells. Phase-contrast microscopy images of the wounded area of Mv1Lu (A) and HaCaT (B) cells were taken under control conditions and compared to images of the cells treated with 10 ng/mL EGF for 19 h in serum-free conditions. Representative micrographs show the extent of scratch gap and closure obtained after wounding with a 20 &#181;L micropipette tip. Magnification = 10x; Scale bar = 100 &#181;m. Cell migration was quantified and represented as variation of the area differences between treatments and control samples for each assay (arbitrary units; AU). The plot is representative of eight independent measurements for each condition. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56799/56799fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56799/56799fig2.jpg" /> 
Figure 2. Actin filaments and c-JUN expression changes promoted by EGF in migrating HaCaT cells. (A) Overnight 10 ng/mL EGF treatment promotes profound changes in the conformation of actin filaments in migrating HaCaT cells. Cells at the migration front of control conditions portray a tightly structured actin filaments cytoskeleton, while cells treated with EGF show poor actin filaments organization at the migration front. Magnification = 63x; Scale bar = 20 &#181;m. F-Actin is color-coded red, and Hoechst is represented by a blue color. (B) When immuno-fluorescence is used in combination with LSM imaging to study c-JUN expression after 10 ng/mL EGF treatment on migrating HaCaT cells, tiling compositions reveal spatial expression patterns. Images of c-Jun fluorescence (green) were converted into pseudo-color to show the intensity of c-Jun staining. The color rainbow scale represents fluorescence intensity for c-Jun, corresponding to colormaps on the right panels for EGF and Control. Co-staining with phalloidin (red) and Hoechst-33258 (blue) was used to show cell structure and nuclei, respectively. Images were taken by a confocal microscope. Note the increasing expression levels of nuclear c-Jun in areas nearing the migrating front. Scale bar = 40 &#181;m. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56799/56799fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

microscopy-based-methods-for-assessment-epithelial-cell-migration

Universidad San Sebastian

Research

JoVE Journal

Medicine

9.2K Views.  IMIB-Arrixaca. 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.

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

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the beginning and end of an experiment using a phase-contrast microscope, and the migration abilities of cells are evaluated by the closure of wounds. However, cell movement is a dynamic phenomenon, and a conventional method does not allow for tracking single-cell movement. To improve current wound-healing assays, we use live-cell imaging techniques to monitor cell migration in real time. This method allows us to determine the cell migration rate based on a cell tracking system and provides a clearer distinction between cell migration and cell proliferation. Here, we demonstrate the use of live-cell imaging in wound-healing assays to study the different migration abilities of breast epithelial cells influenced by the presence of TIP60. As cell motility is highly dynamic, our method provides more insights into the processes of wound healing than a snapshot of wound closure taken with the traditional imaging techniques used for wound-healing assays.

Here, we present the real-time monitoring of cell migration in a wound-healing assay using TIP60-depleted MCF10A breast epithelial cells. The implementation of live-cell imaging techniques in our protocol allows us to analyze and visualize single-cell movement in real time and across time.

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the ...

Cancer Research

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an &#34;in-incubator&#34; video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

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

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

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

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

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

Environment

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

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

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

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

Bioengineering

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

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

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

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

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

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.

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.

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 ...

Immunology and Infection

In vitro Cell Migration and Invasion Assays

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

In vitro Cell Migration and Invasion Assays

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

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

Biology

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

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

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

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

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

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

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

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

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

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