Research reported in this publication was supported by the National Institute on Minority Health and Health Disparities of the National Institutes of Health under Award Number U54MD012388. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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<li>Agusa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+exposure+to+arsenic+from+drinking+water+in+Vietnam%5BTitle%5D)+AND+%22Science+of+the+Total+Environment%22%5BJournal%5D)">Human exposure to arsenic from drinking water in Vietnam.</a> Science of the Total Environment. , 488-489, 562-569 (2014).</li>
<li>Ayotte, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Factors+affecting+temporal+variability+of+arsenic+in+groundwater+used+for+drinking+water+supply+in+the+United+States%5BTitle%5D)+AND+%22Science+of+the+Total+Environment%22%5BJournal%5D)">Factors affecting temporal variability of arsenic in groundwater used for drinking water supply in the United States.</a> Science of the Total Environment. , (2014).</li>
<li>Dummer, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Geostatistical+modeling+of+arsenic+in+drinking+water+wells+and+related+toenail+arsenic+concentrations+across+Nova+Scotia%2C+Canada%5BTitle%5D)+AND+%22Science+of+the+Total+Environment%22%5BJournal%5D)">Geostatistical modeling of arsenic in drinking water wells and related toenail arsenic concentrations across Nova Scotia, Canada.</a> Science of the Total Environment. , (2014).</li>
<li>Sorg, T. J., Chen, A. S., Wang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Arsenic+species+in+drinking+water+wells+in+the+USA+with+high+arsenic+concentrations%5BTitle%5D)+AND+%22Water+Research%22%5BJournal%5D)">Arsenic species in drinking water wells in the USA with high arsenic concentrations.</a> Water Research. 48, 156-169 (2014).</li>
<li>Beamer, P. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Association+of+Children%27s+Urinary+CC16+Levels+with+Arsenic+Concentrations+in+Multiple+Environmental+Media%5BTitle%5D)+AND+%22International+Journal+of+Environmental+Research+and+Public+Health%22%5BJournal%5D)">Association of Children's Urinary CC16 Levels with Arsenic Concentrations in Multiple Environmental Media.</a> International Journal of Environmental Research and Public Health. 13, (2016).</li>
<li>Pomari, E., Dalla Valle, L., Pertile, P., Colombo, L., Thornton, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracrine+sex+steroid+synthesis+and+signaling+in+human+epidermal+keratinocytes+and+dermal+fibroblasts%5BTitle%5D)+AND+%22The+FASEB+Journal%22%5BJournal%5D)">Intracrine sex steroid synthesis and signaling in human epidermal keratinocytes and dermal fibroblasts.</a> The FASEB Journal. 29 (2), 508-524 (2015).</li>
<li>Gotsulyak, Y., Kosach, R., Cherednyk, V., Tykhonkova, O., Khoruzhenka, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+cell+motility+evaluation+in+scratch+assay%5BTitle%5D)+AND+%22Biopolymers+and+Cell%22%5BJournal%5D)">Optimization of cell motility evaluation in scratch assay.</a> Biopolymers and Cell. 30, 223-228 (2014).</li>
<li>Goetsch, K. P., Niesler, C. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+the+scratch+assay+for+in+vitro+skeletal+muscle+wound+healing+analysis%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">Optimization of the scratch assay for in vitro skeletal muscle wound healing analysis.</a> Analytical Biochemistry. 411, 158-160 (2011).</li>
<li>Harishkumar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Revealing+the+mechanism+of+in+vitro+wound+healing+properties+of+citrus+tamurana+extract%5BTitle%5D)+AND+%22BioMed+Research+International%22%5BJournal%5D)">Revealing the mechanism of in vitro wound healing properties of citrus tamurana extract.</a> BioMed Research International. 13, Article ID 963457, 8 pages (2013).</li>
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</ol>

The authors have nothing to disclose.

Application of this assay provides a high-throughput and timely evaluation of cellular migration and may directly translate to complex physiological systems5,6,7. For this reason, the proposed bench-top model has been tuned and practiced to evaluate the effects of various therapeutic agents and environmental toxins on wound healing6. The concentrations of arsenic used in the current study were deemed environmentally relevant through extensive literature search of known exposure levels, and from retrospective review of various data bases14,15,16,17,18,19,20,21. Use of this in vitro scratch assay demonstrated that exposure to an arsenic concentration within the high, but environmentally relevant range, delayed wound closure (cellular migration).
This bench top model was also employed to isolate and study an individual cell line (fibroblast) to further understand how fibroblast migration contributes to wound healing outcomes. Furthermore, it is difficult to study the functions of an individual cell line in complex in vivo systems with many other cells and tissues present that can interfere with the analysis. Additionally, the assay provides a means of achieving semi-quantitative percent wound closure data that can be used to target specific cellular mechanisms through which compounds may influence wound healing. For example, cells used in the scratch assay can be collected and stored in a desired reagent and used in gene expression (signal mRNA) or protein expression assays22. This information can be useful to the investigator if they seek to understand what signals or proteins may be largely contributing to changes in cellular migration in the presence of varying treatments (i.e., arsenic)22.
Human neonatal dermal fibroblasts were selected for this work based on their prominent role in wound healing. However, this scratch assay protocol has been implemented using various cell types and for a range of research purposes. For example, the scratch assay has been adopted into the field of oncology for studying migration inhibition of chemotherapy drugs23; for skeletal muscle cell migration, proliferation, and diffusion24; to study the effect of plant extracts on cellular migration25; and to understand how keratinocyte migration and proliferation contribute to wound healing9. Additionally, a previous paper by Pinto et al. evaluated the effects of uranium (U) and platelet-rich plasma (PRP) on hDFn migration using the scratch assay6. The purpose of this work was to understand the extent to which this assay could test the effect of an agent thought to slow cellular migration (U), as well as an agent thought to speed up cellular migration (PRP). As can be seen in the aforementioned work and research by other investigators, application of the scratch assay has high potential for use in various experimental models26. In spite of the detail provided within the methods, variance should be expected between investigators and experiments. For example, cellular migration rates will likely fluctuate based on the cell line in use, in addition to the required micronutrients needed for unique cell types. Many of the important observations to aid in cell yield and viability are listed as NOTEs throughout this protocol. However, it is strongly recommended that investigators follow manufacturer&#39;s recommendations for optimal growth conditions, and to use this method as supplementary instruction for general cell culture work.
Although the scratch assay is extremely versatile for study of cellular activity; unforeseen biases may arise in measuring techniques. For example, when using ImageJ to manually trace cell migration fronts, variation may exist among users, which can limit accuracy and representative data collection. It should also be noted that migration fronts should be read blind to the treatment to minimize user bias. Furthermore, manually identifying migration fronts may result in miscalculation of average distance traveled due to pseudopod formation by cells and their ability to reach out to form focal adhesions, which can be visually misleading. In like manner, the use of fluorescent &#34;cell tracker&#34; dyes on cell surface or nuclear proteins to detect migration may unintentionally fix cells resulting in inaccuracies when measuring cellular migration over time. It is recommended that appropriate positive and/or negative controls be enrolled within the experiment to completely asses the effect of treatment on the cell lines. Practice of this assay must be completed in recognition of its limitations. This assay does not guarantee that cellular migration results obtained from in vitro experimentation will directly translate to in vivo outcomes. Wound healing and cellular migration in a complex living system involves a multitude of cell types and molecular signals; each of which has the potential to influence healing responses.
In summary, the scratch assay has been found to provide high-throughput screening of cellular migration in response to changing environments6,25. We specifically utilized this protocol to successfully detect changes in cellular migration as a result of arsenic exposure at various environmentally relevant doses. These data have provided justification to use the scratch assay to further study and evaluate the mechanistic pathways of cellular migration in this assay and how these pathways are altered by arsenic exposure.

Wound healing consists of a complex series of overlapping steps that involve various cell types, signaling molecules, and extracellular matrix components1. Together, these components work in concert to achieve wound resolution or repair, which ultimately leads to the formation of a protective fibrotic scar depending on the degree of trauma1. To capture the individual processes and functions of wound healing in one experiment is difficult; individual steps within the wound healing process need to be identified and tested separately. Among the many steps of wound healing, the process of cellular migration has been considered critical when evaluating healing rates2. Cellular migration can be observed in all phases of wound healing, and thus necessitates further understanding of how alterations to cell migration can impact wound closure. For example, cellular migration is seen in both early and late phase inflammation, with blood coagulation and platelet aggregation at the wound site3. These events prevent excessive blood loss by forming a temporary blockage to fill wounded areas devoid of tissue3. Platelets in circulation will synthesize and secrete vasoactive mediators along with chemotactic factors, which together signal for inflammatory leukocyte migration (white blood cells) to the wounded tissue for repair4. Re-epithelialization occurs within hours of tissue injury and involves covering the wound site through activity and migration of epithelial keratinocytes to prevent further contamination or invasion of foreign particles to the wound site2. These examples capture only a few of the many cell migration functions associated with wound healing.
The scratch assay has been described and used to better understand cell migration and its many roles in wound healing5,6. This assay is considered simplistic, provides high-throughput, and enables the investigator to collect representative cell migration data. Migration assays have successfully demonstrated that certain compounds may accelerate or slow down the rate of cellular migration6. Furthermore, these assay results provide translational physiologic information that can be used to formulate target treatments, dosing concentrations, and genes of interest prior to in vivo experimentation. The assay requires basic cell culture techniques and supplies, a cell line of choice, and imaging technology to capture movement over time or movement in response to treatments.
The assay can be readily manipulated or tailored to suit the needs of the investigator. However, there are four basic questions to consider prior to experimentation. What general or specific question(s) is/are the investigator(s) trying to answer? This holds true for most hypotheses driven research; however, it is critical to this assay because it will determine many of the parameters needed to accurately and completely answer the question(s). What cell line or cell lines (if multiple) should be used to represent the physiologic system being studied? For example, in a cutaneous wound healing study, a dermal fibroblast5,6,7, stem cell8, or epidermal keratinocyte9 might be considered to represent cell populations that are normally found in abundance in skin tissue10. Alternatively, neutrophils, macrophages or other inflammatory cells could be evaluated in this system to represent cell migration of other components of wound healing within skin tissue10. Additionally, identifying the specific cell line being evaluated will help to identify which reagents are optimal to use to promote cell metabolic activity. How will cell migration be tracked/imaged? There are a number of techniques used to observe cell migration within a plate or flask. For example, dyeing or fluorescently tagging cells and using fluorescence or confocal imaging to track cells provides a strong contrast, which allows the investigator to clearly define cellular vs. non-cellular locations and cellular movement11. Another common technique, described in this study, is the use of inverted light microscopy with phase contrast6,7. This alternative to cell dyes and fluorescence allows the user to live track cells without having to terminally fix cells prior to imaging and also allows for capturing images of the same individual wells containing wounded monolayers of cells across time points. What outcome measures will be used for analysis? Using the images gathered throughout the study, data can be generated in which changes to cellular migration rates can be understood. In our lab, microscopic images captured on the inverted light microscope are uploaded to image analysis software and the percent wound closure and summed area under the curve (AUC) are calculated.
We will highlight the specific use of this assay to elucidate changes to dermal fibroblast migration in the presence of a known environmental contaminant, arsenic. Arsenic is a naturally occurring metalloid found within the earth&#39;s crust. Various locations have been found with elevated levels of arsenic, which poses a risk to individuals who live near these locations. As a result, food and water sources have been shown to have increased levels of arsenic contamination, which increases the likelihood for individuals to become exposed to arsenic. Environmental arsenic exposure has been shown to have long-term health consequences in human populations above or even below the current United States Environmental Protection Agency (USEPA) Max Contaminant Level (MCL) of 10 ppb (10 &#181;g/L)12,13. Although the USEPA has set this MCL and deemed it safe for drinking limits, arsenic concentrations that exceed this limit are not uncommon in water sources worldwide. In the Southwestern United States, numerous ground-water, well water, and springs have been documented to contain concerning levels of arsenic14. For example, in Verde Valley, AZ, Montezuma Well contains 210 &#181;g/L&#160;of arsenic15. Groundwater around the Verde River, AZ contains 16 &#181;g/L&#160;of arsenic on average, with peak values reaching 1.3 mg/L15,16. Notably, arsenic concentrations in many wells in the Verde River water shed exceed 50 &#181;g/L15,16. With this knowledge, environmentally relevant arsenic concentrations were selected that range from below the MCL at 0.1 &#181;M (7.5 ppb), to above the MCL at 10 &#181;M (750 ppb) in the current study14,15,16,17,18,19,20.
The information gathered from these experiments will be used as an early indicator of potential changes to cellular migration rates in an in vivo wound contaminated with arsenic. Afterward, signaling molecules can be selected based on their role in facilitating wound healing and cellular migration, and future quantitative polymerase chain reaction (qPCR) based gene expression experiments may be set up upon completion of the current studies. If migration rates in this assay change in the presence of arsenic, specific gene targets involved in cellular migration may be the target for deleterious effects of arsenic, and thus may be the mechanism of disruption.

<table><tbody><tr><td>Class II A/B3 Biological Safety Cabinet</td><td>Forma Scientific</td><td>15201-816</td><td></td></tr><tr><td>Nitrile Gloves</td><td>Kimberly-Clark</td><td>55082</td><td></td></tr><tr><td>Water Bath</td><td>Precision</td><td>Model 182</td><td></td></tr><tr><td>Inverted Microscope</td><td>Leica</td><td>Q080318</td><td></td></tr><tr><td>CPR Centrifuge</td><td>Beckman</td><td>349702</td><td></td></tr><tr><td>Analytical Scale</td><td>Ohaus</td><td>I093 1125062111P</td><td></td></tr><tr><td>Weighting paper</td><td>VWR</td><td>H351125781212A</td><td></td></tr><tr><td>Biohazard bags</td><td>Fisherbran</td><td>01-830A</td><td></td></tr><tr><td>Water Jacketed CO&lt;sub&gt;2 &lt;/sub&gt;Incubator</td><td>Thermo Scientific</td><td>309739-31053</td><td></td></tr><tr><td>Hanks Balanced Salt Solution</td><td>Gibco</td><td>14175-095</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Gibco</td><td>10566-016</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>26140-079</td><td></td></tr><tr><td>Automatic Pipettor</td><td>Drummond</td><td>140685</td><td></td></tr><tr><td>p200 Pipette</td><td>Gilson</td><td></td><td></td></tr><tr><td>p20 Pipette</td><td>Gilson</td><td></td><td></td></tr><tr><td>Denominator</td><td>Fisher Scientific</td><td>D59707</td><td></td></tr><tr><td>Trypan Blue Stain</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>T75 tissue flask</td><td>Corning</td><td>430725U</td><td></td></tr><tr><td>15 mL Conical Tube</td><td>Fisherbrand</td><td>05-539-5</td><td></td></tr><tr><td>50 mL Conical Tube</td><td>VWR</td><td>21008-178</td><td></td></tr><tr><td>5 mL Pipette</td><td>Fisherbrand</td><td>13-678-11D</td><td></td></tr><tr><td>10 mL Pipette</td><td>Fisherbrand</td><td>13-678-11E</td><td></td></tr><tr><td>Tissue Marker</td><td>Securline</td><td>92167</td><td></td></tr><tr><td>Wipes</td><td>Kintech</td><td>34155</td><td></td></tr><tr><td>70% Ethanol</td><td>Fisher</td><td>63-67-0</td><td></td></tr><tr><td>Non-Filter Pipet Tips</td><td>Fisherbrand</td><td>16160210</td><td></td></tr><tr><td>Bleach</td><td>Great Value</td><td>220-04</td><td></td></tr><tr><td>96-well</td><td>Falcon</td><td>353915</td><td></td></tr><tr><td>Cryovial Rack</td><td>Nalgene</td><td>5030-0505</td><td></td></tr><tr><td>Hemacytometer</td><td>Gizmo Supply</td><td>B00SCOGY56</td><td></td></tr><tr><td>TrypLE Express</td><td>Gibco</td><td>12605-028</td><td></td></tr><tr><td>Conical Tube Rack</td><td>n/a</td><td>n/a</td><td></td></tr><tr><td>12-well plate</td><td>Corning</td><td>3598</td><td></td></tr><tr><td>Waste Beaker</td><td>n/a</td><td>n/a</td><td></td></tr><tr><td>1 mL Pipette</td><td>Fisherbrand</td><td>13-678-11B</td><td></td></tr><tr><td>Straight Edge Ruler</td><td>n/a</td><td>n/a</td><td></td></tr><tr><td>Human neonatal dermal fibroblasts (hDFn)</td><td>lot: 1734, 2902</td><td>106-05n</td><td></td></tr><tr><td>Rstudio Statistical Software</td><td></td><td>ver: 3.5.1</td><td></td></tr><tr><td>Image J</td><td>NIH</td><td>ver: 1.8.0_112</td><td></td></tr></tbody></table>

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.

Sample sizes (n) for all treatment groups was n = 28. Cells were successfully cultured following aseptic techniques and lab protocols (Figure 1). Uniform scratches were observed across all treatment groups with the mean scratch width measuring 0.8 mm 0.4 mm (Figure 2). Control groups had an average summed area under the curve value of 1,355.83; 0.01 µM As treatment groups had an average summed area under the curve value of 1,366.21; 0.1 µM As treatment groups had an average summed area under the curve value of 1,326.24, 1.0 µM As treatment group had an average summed area under the curve value of 1,295.10; and finally the 10 µM As treatment group had an average summed area under the curve value of 535.12, which was statistically lower compared to all other groups (p &lt; 0.05) (Figure 3). R-program for statistical analysis was used to run a one-way ANOVA with a Tukey post hoc adjustment to determine statistical differences among treatment groups (p &lt; 0.05).
<img alt="Cellular growth over 20 hours with arsenic; microscope images; 0, 1.0, 10 μM As concentrations." class="xfigimg" src="/files/ftp_upload/58838/58838fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Representative scratch assay images over a 20 h period. Images A-D represent cellular migration of control cells; images E-H represent cellular migration of cells contaminated with 1.0 µM As; images I-L represent cellular migration of cells contaminated with 10 µM As. From these images, it is clear that cellular migration is slowed in the presence of arsenic. The control images show a completely "healed" scratch after 20 h, while the other two As-treatment groups were not "healed". The 10 µM As treatment inhibited complete closure altogether after 24 h. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58838/58838fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="AUC arsenic dose response chart, arsenic concentration vs. summed AUC, bar graph analysis." class="xfigimg" src="/files/ftp_upload/58838/58838fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Arsenic slows cellular migration in the scratch assay. Images were captured over a 24 h period to observe changes to cellular migration rates in the presence of varying concentrations of arsenic. Raw images were analyzed using an automated software (e.g., in MATLAB), which initially measured wound width, then calculated percent closure and finally area under the curve (AUC). Error bars represent standard error of the mean. The 10 µM As treatment group statistically slowed cellular migration of human dermal fibroblasts neonatal (hDFn), shown by a statistically lower AUC value, compared to all other treatment groups using a one-way ANOVA with a Tukey post hoc adjustment (p&lt;0.05). Statistical differences are represented by the letters "A" and "B", which were obtained through using the Compact Letter Display method available in R-program. Treatments that do not share a common letter are statistically significant from each other (p&lt;0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58838/58838fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration at JoVE.com

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.

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 ...

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22.9K Views.  Northern Arizona University. Cellulaire migratie is een kritisch wondgenezingsproces dat de uitkomst van een genezende wond drastisch kan veranderen. Dit protocol stelt onderzoekers in staat om het effect van verschillende verbindingen op cellulaire migratie tijdens wondgenezing beter te begrijpen. Wat vaak kan leiden tot gerichte behandeling strategieën voor klinische geneeskunde.De scratch test is een kosteneffectieve en high-throughput aanpak voor het genereren van zinvolle wondgenezingsgegevens voorafgaand aa...

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

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 ...

Cancer Research

Using the Dot Assay to Analyze Migration of Cell Sheets

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move within tissues in a tightly orchestrated manner. During tumor development, this equilibrium is disturbed, and tumor cells leave the epithelium of origin to invade the local microenvironment, to travel to distant sites, and to ultimately form metastatic tumors at distant sites. The dot assay is a simple, two-dimensional unconstrained migration assay, to assess the net movement of cell sheets into a cell-free area, and to analyze parameters of cell migration using time-lapse imaging. Here, the dot assay is demonstrated using a human invasive, lung colony forming breast cancer cell line, MCF10CA1a, to analyze the cells' migratory response to epidermal growth factor (EGF), which is known to increase malignant potential of breast cancer cells and to alter the migratory phenotype of cells.

Cell migration is essential for development, tissue maintenance and repair, and tumorigenesis, and is regulated by growth factors, chemokines, and cytokines. This protocol describes the dot assay, a two-dimensional, unconstrained migration assay to assess the migratory phenotype of attached, cohesive cell sheets in response to microenvironmental cues.

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move ...

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.

The capacity to migrate is a key function of many different cell types, including mesenchymal stromal cells (MSCs). However, quantifying alterations in migratory capacity after damage is challenging. This protocol describes an easily adaptable migration assay that uses rigorous statistics to quantify changes in MSC migratory capacity after damage.

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

Developmental Biology

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 ...

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

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.

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.

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

Medicine

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.

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for ...

Biology

A Cost Effective and Adaptable Scratch Migration Assay

Cell migration is a key component in both physiological and pathological events. Normal cell migration is required for essential functions such as development and mounting an immune response. When a defect or alteration occurs with the cell migration process, it can have detrimental outcomes (i.e., cancer metastasis, wound healing, and scar formation). Due to the importance of cell migration, it is necessary to have access to a cell migration assay that is affordable, adaptable, and repeatable. Utilizing the common scratch migration assay, we have developed a new approach to analyzing cell migration that uses general laboratory equipment. The method described uses visual markers that allow for recapturing specific areas of interest without the use of time-lapse microscopy. In addition, it provides flexibility in the experimental design, ranging from altering the migration matrix substrate to the addition of pharmacological modifiers. Furthermore, this protocol outlines a way to account for the area of cell migration, which is not considered by several methods when examining cell migration. This new approach offers a scratch migration assay to a larger audience and will provide greater opportunity for researchers to examine the physiological and pathophysiological impact of cell migration.

We present a cost-effective method to the scratch migration assay that provides a new approach for determining cell migration without the use of equipment-intensive methods. While fibroblasts were used in this protocol, it can be adapted and utilized to study additional cell types and influences on cell migration.

Cell migration is a key component in both physiological and pathological events. Normal cell migration is required for essential functions such as ...

Study of Angiogenesis Using Scratch Wound Migration and Spheroid Sprouting Assays

Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A detailed understanding of the underlying molecular mechanisms and reliable screening models are essential for targeting diseases effectively and developing new therapeutic options. Several in vitro assays have been developed to model angiogenesis, capitalizing on the opportunities a controlled environment provides to elucidate angiogenic drivers at a molecular level and screen for therapeutic targets.
This study presents workflows for investigating angiogenesis in vitro using human umbilical vein endothelial cells (HUVECs). We detail a scratch wound migration assay utilizing a live cell imaging system measuring endothelial cell migration in a 2D setting and the spheroid sprouting assay assessing endothelial cell sprouting in a 3D setting provided by a collagen matrix. Additionally, we outline strategies for sample preparation to enable further molecular analyses such as transcriptomics, particularly in the 3D setting, including RNA extraction as well as immunocytochemistry. Altogether, this framework offers scientists a reliable and versatile toolset to pursue their scientific inquiries in in vitro angiogenesis assays.

Author Spotlight: Investigating Angiogenesis Through Challenges and Innovations in Assay Development

This study presents the two-dimensional (2D) scratch wound migration assay and the three-dimensional (3D) spheroid sprouting assay, along with their respective downstream analysis methods, including RNA extraction and immunocytochemistry, as suitable assays to study angiogenesis in vitro.

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A ...

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

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