This work is supported by the US Army Medical Research Award #81XWH-16-1-0710, University of Mississippi School of Pharmacy and the Department of BioMolecular Sciences.

1. General cell culture
<ol>
	<li>Culture cardiac fibroblasts in Dulbecco&#8217;s Modified Eagles Medium (DMEM) containing 1 g/L glucose, sodium pyruvate, L-glutamine, and supplemented with 14.2 mM NaHCO3, 14.9 mM HEPES, 15% heat-inactivated fetal bovine serum (FBS), 2% L-glutamine, and 0.02% antimicrobial reagent (see Table of Materials) and maintained in CO2 incubator at 37 &#176;C.</li>
	<li>Culture cardiac fibroblasts until 90-95% confluency is reached at passage 0 (P0). At this point the fibroblasts are ready to be split into a 48-well plate, which is used for the migration assay.</li>
</ol>
2. Preparation of the migration plate
<ol>
	<li>Prepare a 48-well cell culture plate by drawing a line, using a yellow or light-colored permanent marker, down the center of the well. Then, draw three hash marks dividing the well into three separate areas of interest. These sections will be used for imaging. 
	NOTE: Using a light color permanent marker will allow for easy imaging of migrating cells. Dark markers such as black or blue will prevent visualization of migrating cells.</li>
	<li>If assessing migration on a substrate (e.g., collagen), coat the well at this time following instructions and concentrations used for the specific substrate.</li>
	<li>Plate ~15,000-20,000 cardiac fibroblasts, P1, into each well and culture cells, under normal culturing conditions (conditions listed in previous section), until they reach 90-95% confluency. Confluency should be reached between 24-48 hours. 
	NOTE: When setting up the migration plate make sure to include a positive control (a known migratory cell, such as 3T3 cells18), a negative control (unscratched cells) and a blank well.</li>
</ol>
3. Scratch migration assay &#38; fixation
<ol>
	<li>Once cells reach 90-95% confluency, remove the media and scratch along the drawn line using a sterile P200 pipette tip. 
	NOTE: A P200 pipette tip is the common pipette tip sized used with the scratch assay10,14,19. Also, only make one pass with the P200 tip as more than one attempt may result in multiple scratch lines.</li>
	<li>Rinse the well with low serum media (1.5% FBS) to remove any unattached cardiac fibroblasts.</li>
	<li>Add 500 &#181;L of low serum media to each well. If using any pharmacological agents, add them at this time. 
	NOTE: Low serum media is used because it allows/promotes cell migration to occur and prevents fibroblasts from proliferating which could skew the results of the migration assay20. For this protocol, 1.5% FBS was used.</li>
	<li>Capture 0 h images before incubating the fibroblasts in a 5% CO2 incubator at 37 &#176;C for 24 h.
	<ol>
		<li>Capture 0 h images using an inverted microscope with a 20x objective. Take two 0 h images per well. This will allow for a more complete coverage of the line of migration.</li>
		<li>Using the markings (line and dashes), position the well to capture the top half of the line of scratch. Avoid imaging the middle dash to ensure that the same area of migration is not imaged twice, which could skew results.</li>
		<li>Use imaging software (Table of Materials) to capture 0 h image #1. Then move the plate to position the bottom half of the well/line of scratch in view of the camera. Again, avoid capturing the middle dash. Once in place, capture 0 h image #2 (Figure 1). 
		NOTE: Avoiding the middle dash in both 0 h images will prevent capturing areas of migration twice which if done could produce repeating results and skew the data.</li>
	</ol>
	</li>
	<li>After incubating for 24 h, remove the media from the well, and wash the well with 1x nonsterile PBS (henceforth all 1x PBS used is nonsterile).</li>
	<li>In the fume hood, add 500 &#181;L of 4% paraformaldehyde to the well and incubate for 10 minutes at room temperature (RT).</li>
	<li>Remove paraformaldehyde and wash 3x with 1x PBS for 5 min each at RT.</li>
	<li>Once cells are washed, proceed to capturing 24 h images or add 1x PBS to each well and place at 4 &#176;C until a later time. Cells can stay at 4 &#176;C for 1-2 weeks until ready to image.
	<ol>
		<li>Permeabilize the cells by adding 300 &#181;L of permeabilizing solution (1x PBS and 0.01% triton X-100) to each well. Incubate cells/plate with gentle, continual rocking for 30 min at RT.</li>
		<li>Remove permeabilizing&#160;solution and add 1% Coomassie Brilliant Blue stain (3% Coomassie Brilliant Blue, 10% acetic acid, 45% methanol, and 45% dH2O) for 10 min with gentle, continual rocking at RT. 
		NOTE: If plates are coated with an extracellular matrix substrate make sure to test a coated plate with Coomassie staining before conducting migration assay. Figure 2 demonstrates that a matrix substrate can be used with this assay and not impact visualization of cells.</li>
		<li>Wash wells 3x with 1x PBS at RT with continual rocking for 5 min each.</li>
		<li>After washes, added 300 &#181;L of 1x PBS and capture 24 h images.</li>
		<li>Capture 24 h images by aligning the well into the same position that was used to capture the 0 h images. In order to accomplish this, open the previously captured 0 h images and using the marking made with the permanent marker, align the well into the same position. The line down the middle and additional dash marks should allow close alignment between the 24 h image and the 0 h image (Figure 1).</li>
	</ol>
	</li>
</ol>
4. Preparation of 0 h and 24 h images
<ol>
	<li>Open 0 h and 24 h images taken of the same well and position in imaging software (Table of Materials).</li>
	<li>Create a new layer on the 0 h image. Then, click the new layer and change the name of the layer to &#8220;line layer&#8221; by double clicking on the text. 
	NOTE: This layer will be referred to as line layer from this point forth.</li>
	<li>Click the Brush Tool (brush icon) and set the size at 10 px and the color to red.</li>
	<li>With the line layer selected, draw two, separate lines that outline the area of the scratch. The lines should not touch any cells due to these lines marking the area of migration.</li>
	<li>Click the Move Tool (arrow icon) and then press down Ctrl button and click on both the line and background layers.</li>
	<li>Click in the center of the 0 h image and drag both these layers to the middle of the 24 h image.</li>
	<li>Now using the 24 h image, click on the 0 h background layer and change the opacity to 50%.</li>
	<li>Holding the Ctrl button, click on both the 0 h background and line layer and then free transform the layers (Edit&#62;Free Transform). Using free transformation, align the 0 h background and line image to the 24 h background image. This step results in the overlaying of the 0 h and 24 h image, which is necessary to position the lines that mark the area of migration in the correct position on the 24 h image.</li>
	<li>After successful overlaying of the 0 h background and line layers onto the 24 h background image, delete the 0 h background layer. Delete by clicking the 0 h background layer, then right click, and click delete layer.</li>
	<li>Deleting the 0 h background image will leave just the line and the 24 h background layer (now referred to as migration image). The line layer will indicate the area of migration/scratch on the 24 h image and can be used for determining the number of migrating cells.</li>
	<li>Save as the new migration image as both a photoshop file and a TIFF/JPEG. NOTE: a representative figure that depicts this process is presented in Figure 3.</li>
</ol>
5. Counting the number of migrating cells
<ol>
	<li>Open the migration image. This can be done in a program that accepts TIFF/JPEG files (Table of Materials).</li>
	<li>Count the number of cells that are located in between as well as touching the two red lines.</li>
	<li>Record the number of migrating cells per image. There will be two migrating images per well, and these values should not be combined until after the values have been corrected for area of migration (detailed in next section).</li>
</ol>
6. Determine area of migration
<ol>
	<li>Open the 24 h image that contains the lines of migration in the imaging software program in section 5 (Table of Materials).</li>
	<li>Save As this image as &#8220;Migration Area Image&#8221;.</li>
	<li>After saving, click the background layer, right click, and click delete layer. This should leave only the scratch lines on the image (Figure 3).</li>
	<li>Using the Brush Tool (brush icon) fill in the area between the lines of scratch. Match the color of the brush with the color used to draw the lines for migration.</li>
	<li>Change the image to a grayscale to generate a black and white image (Image&#62;Mode&#62;Grayscale) and then save the image (Figure 4).</li>
	<li>Start the analysis software (Table of Materials) and then open the &#8220;Area of Migration&#8221; file within analysis software.</li>
	<li>To determine the area of the line of migration, click Image&#62;Adjust&#62;Threshold. The black migration area will turn red and the percent area will be indicated in the Threshold box. The area will be reported as the percent of area the line of migration covers compared to the entire area captured in the image.</li>
	<li>Record the percent area for the line of migration and make sure that this value is paired with the number of migrating cells for the same image.</li>
</ol>
7. Data analysis
<ol>
	<li>Normalize the number of migrating cells to the percent area of the scratch. Divide the number of migrating cells by the percent area of the line of migration (# migrated cells % Area of migration). Do this per image and not an average based on migration per well.</li>
	<li>After normalizing values per image, add the values of the two images, which represents an individual well. This value will be used for graphical and statistical analysis. If the experimental design contained multiple replicates. Average replicate values before statistical analysis.</li>
</ol>

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<li>Stewart, J. A., Massey, E. P., Fix, C., Zhu, J., Goldsmith, E. C., Carver, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Temporal+alterations+in+cardiac+fibroblast+function+following+induction+of+pressure+overload%5BTitle%5D)+AND+%22Cell+and+tissue+research%22%5BJournal%5D)">Temporal alterations in cardiac fibroblast function following induction of pressure overload.</a> Cell and tissue research. 340 (1), 117-126 (2010).</li>
<li>Darby, I. A., Laverdet, B., Bonté, F., Desmoulière, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibroblasts+and+myofibroblasts+in+wound+healing%5BTitle%5D)+AND+%22Clinical%2C+Cosmetic+and+Investigational+Dermatology%22%5BJournal%5D)">Fibroblasts and myofibroblasts in wound healing.</a> Clinical, Cosmetic and Investigational Dermatology. , 7(2014).</li>
<li>Kramer, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+cell+migration+and+invasion+assays%5BTitle%5D)+AND+%22Mutation+Research+-+Reviews+in+Mutation+Research%22%5BJournal%5D)">In vitro cell migration and invasion assays.</a> Mutation Research - Reviews in Mutation Research. 752 (1), 10-24 (2013).</li>
<li>Even-Ram, S., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+migration+in+3D+matrix%5BTitle%5D)+AND+%22Current+Opinion+in+Cell+Biology%22%5BJournal%5D)">Cell migration in 3D matrix.</a> Current Opinion in Cell Biology. (17), 524-532 (2005).</li>
<li>Valster, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+migration+and+invasion+assays%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Cell migration and invasion assays.</a> Methods. 37, 208-215 (2005).</li>
<li>Pijuan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+cell+migration%2C+invasion%2C+and+adhesion+assays%3A+From+cell+imaging+to+data+analysis%5BTitle%5D)+AND+%22Frontiers+in+Cell+and+Developmental+Biology%22%5BJournal%5D)">In vitro cell migration, invasion, and adhesion assays: From cell imaging to data analysis.</a> Frontiers in Cell and Developmental Biology. 7, 1-16 (2019).</li>
<li>Liang, C. C., Park, A. Y., Guan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+scratch+assay%3A+a+convenient+and+inexpensive+method+for+analysis+of+cell+migration+in+vitro%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro.</a> Nature Protocols. 2 (2), 329-333 (2007).</li>
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<li>Lipton, A., Klinger, I., Paul, D., Holleyt, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Migration+of+Mouse+3T3+Fibroblasts+in+Response+to+a+Serum+Factor%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Migration of Mouse 3T3 Fibroblasts in Response to a Serum Factor.</a> Proceedings of the National Academy of Sciences of the United States of America. 68 (11), (1971).</li>
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The authors have nothing to disclose.

This new approach to the scratch migration assay provides a more accessible method for researchers to examine changes in cell migration. While this assay follows the same procedure for administering a scratch similar to other scratch assays, it does provide a new method for imaging and accurate analysis of cell migration10. Instead of using equipment-intensive methods of time-lapse microscopy and live cell imaging chambers, this method details the use of commonly available lab equipment. Utilizing...

Cell migration is crucial for many physiological as well as pathological events. It is required during development, for mounting an immune response, and for proper wound healing1,2,3. Many of these cell migration events can be triggered by physical or chemical signals. For example, during an immune response, leukocytes will migrate towards a site of injury in response to a chemoattractant2. Additionally, leukocytes will also release cytokines to induce migration of additional immune cells, as well as other cell types, such as fibroblasts, which are involved in the wound healing process, and thus, initiating a multicellular response4. The ability of cells to migrate is essential for proper physiological function; however, when cell migration goes unchecked, it can have an adverse response and contribute to pathological events, such as chronic inflammation, vascular disease, cancer metastasis, and impaired wound healing2,3,4,5,6,7. Impaired wound healing is a common affliction of diabetics due to defects in cell migration, and if these defects are not addressed, it could lead to further complications (e.g., amputation8,9). This study, as well as others, have indicated the need to further understand the process by which cell migration occurs, either under normal physiological or pathological conditions, and it is vital for furthering this field of research. In order to accomplish this, there needs to be migration assays available that are both accessible and affordable to those researchers, who may not possess the equipment needed to conduct these assays.
Currently, there are a variety of migration assays available to examine a wide range of topics regarding cell migration. Both 2D and 3D migration models have been developed, each targeting specific areas impacting cell migration. 3D migration models are typically associated with cell invasion studies and assess the impact of extracellular matrix on cell migration10,11,12, whereas 2D migration assays have a greater range of application and are primarily used to study chemotactic migration, wound healing, and functional changes during cell migration13,14,15,16. Several of these assays require additional equipment, such as Boyden chambers or exclusion rings, which can reduce the availability of these assays to certain researchers. One of the more cost-efficient assays is the scratch assay, which is typically used to assess wound healing and general changes in cell migration14,17. While most laboratories are equipped to conduct a scratch assay, the equipment used to track cell migration tend to either be unavailable or too expensive to purchase. This includes time-lapse microscopy, which requires an inverted microscope and a live imaging system. These expensive pieces of equipment are not commonly accessible to every laboratory. Therefore, this observation highlights the need for a new protocol that allows assessment of cell migration with more readily available equipment.
The protocol presented here provides a new and affordable way to assess cell migration. This method follows the same procedure associated with scratch assays but differs in the analysis of examining cell migration by utilizing equipment more commonly available in a basic sciences laboratory setting. This protocol using common equipment allows for a more accurate determination of cell migration without the use of time-lapse microscopy. In addition to determining migration, this method also accounts for variable factors in the scratch area that has been noted to greatly impact cell migration. Overall, this new protocol for cell migration analysis provides an opportunity for more laboratories to explore and contribute to the field of cell migration.

<table><tbody><tr><td>Adobe All Apps</td><td>Adobe</td><td></td><td>This includes Adobe photoshop which is the imaging software used with this protocol</td></tr><tr><td>Avant Pipette Tips 200ul Binding non-sterile</td><td>MIDSCI</td><td>AVR1</td><td>Tips are autoclaved to sterilize</td></tr><tr><td>AxioCam Erc 5s Camera</td><td>Zeiss</td><td>426540-9901-000</td><td></td></tr><tr><td>Coomassie Brilliant Blue R-250</td><td>Fisher Scientific</td><td>BP101-25</td><td></td></tr><tr><td>Costar Flat Bottom Cell Culture Plates 48 Wells</td><td>Fisher Scientific</td><td>07-200-86</td><td></td></tr><tr><td>DMEM with L-Glutamine, 1g/L glucose and sodium pyruvate</td><td>Fisher Scientific</td><td>MT10014CM</td><td></td></tr><tr><td>Image J</td><td>NIH</td><td></td><td>This is a free software offered by the US government and is the analysis software used with this protocol</td></tr><tr><td>Paraformaldehyde</td><td>Fisher Scientific</td><td>AC416785000</td><td></td></tr><tr><td>Premium US origin fetal bovine serum</td><td>Innovative Research</td><td>IFBS-HU</td><td></td></tr><tr><td>Primocin</td><td>InvivoGEN</td><td>ant-pm-2</td><td>This is the anitmicrobial used for with this procotol to culture cardiac fibroblasts</td></tr><tr><td>Zeiss Primovert Microscope</td><td>Zeiss</td><td>491206-0002-000</td><td></td></tr><tr><td>Zen Blue Edition 2.3 software</td><td>Zeiss</td><td></td><td>software comes with camera purchase</td></tr></tbody></table>

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.

This procedure documents a new approach to studying cell migration that is both cost-effective and easily adaptable for most labs. Many studies have used time-lapse microscopy to assess cell migration, but the equipment required for this method is not readily available to many laboratories. Whereas utilizing lines and dashes for demarcation allows for the ability to recapture specific areas of interest at different time points without the use of expensive equipment (Figure 1 &#38; <strong cl...

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A Cost Effective and Adaptable Scratch Migration Assay

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 ...

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5.3K Views.  University of Mississippi. Dit protocol biedt een nieuwe methode voor een oude techniek, en het zal een nieuwe aanpak voor een groot aantal fundamentele wetenschappelijke onderzoekers. De belangrijkste voordelen van deze techniek zijn het kosteneffectieve karakter en het aanpassingsvermogen ervan om te worden gebruikt door een breder wetenschappelijk publiek. Om een migratieplaat voor te bereiden, gebruikt u een lichtgekleurde permanente markering om een lijn in het midden van de achterkant van elke put van een 48-putp...

Video: A Cost Effective and Adaptable Scratch Migration Assay

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 Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor metastasis. In this assay, cells are grown to form a confluent monolayer and a mechanical wound is created by scratching with a device. Then the migration rate of the cells towards the denuded area can be monitored by imaging. Our 8-channel mechanical wounder is designed to tackle most of the problems associated with the cell migration assay. Firstly, our wounder can be easily sterilized by autoclaving or with common disinfectants. Secondly, the individual adjustable pins allow even contact with the cell culture plate so that sharp and reproducible wounds can be created. Thirdly, the guiding bars on both sides of the wounder ensure consistent wounding position in each well. The use of disposable plastic pipette tips for wounding can further provide better handling of the wounder as well as to minimize cross-contamination. In conclusion, our cell wounder can provide researchers with a user friendly and reproducible device for performing the cell migration assay using the standard 96-well culture plate.

Here, we present a protocol to perform a semi-high throughput cell migration assay on a 96-well cell culture plate. This protocol is a fast, simple and economical method to create consistent scratch wounds on a cell monolayer.

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

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

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 ...

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 ...

A Cost Effective and Adaptable Scratch Migration Assay

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