Danish Council for Independent Research, Technology and Production, grant #4005-00029

The experimental setup has no ethical and legal restriction and was conducted in agreement with Roskilde University. An overview of the experimental set-up can be seen in Figure 1 and the mechanisms of the optical camera in Figure 2.
1. Preparation
<ol>
	<li>Perform all steps in a sterile laminar flow-bench by cleaning the bench and all equipments with 70% ethanol prior to the start of the experiment.</li>
	<li>Place the optical camera in a conventional incubator at 37 &#176;C with 5% CO2 to ensure optimal conditions for the cells.</li>
	<li>Grow HaCaT cells in DMEM media with 10% FBS and 1x antibiotics (penicillin and streptomycin) in a T75 flask.</li>
	<li>Use 1 mL 1x trypsin to detach the cells and sub-cultivate them every 3-4 days, by dispersing it over the cellular layer. Pre-heat to room temperature before use.</li>
</ol>
2. Generating a Monolayer of HaCaT Cells in a 48-wells Microtiter Plate
<ol>
	<li>Remove the medium from the T75 flask with a Pasteur pipette.</li>
	<li>Wash the adherent cells in the flask by adding 5 mL 1x sterile PBS to the T75 flask. 
	Note: Ensure that the PBS is free of calcium and magnesium.</li>
	<li>Circulate the PBS and remove it with a Pasteur pipette.</li>
	<li>Repeat step 2.2-2.3.</li>
	<li>Add 1 mL 1x trypsin to the flask and disperse it across the cell layer.</li>
	<li>Incubate the flask at 37 &#176;C in a conventional incubator until the cells are detached (5-8 min is sufficient for most cell lines).</li>
	<li>View under the microscope if the cells are detached. Once the cells are detached, add 9 mL of the regular culture media with 10% FBS to inactivate the 1x trypsin.</li>
	<li>Count the cell with either a hemocytometer, a coulter-counter or similar. 
	Note: Trypan blue can be used to view dead cells. However, in 1x trypsin HaCaT cells are perfectly round when alive.</li>
	<li>Transfer 7.5 x 104 cells/well in 250 &#181;L 10% FBS media to a round-bottomed 48-well tissue culture plate (standard plate). 
	Note: Assay can also be set up in 6-, 12-, 24-, or 96-well plates, which all can be assayed under the optical microscope.</li>
	<li>Gently move the plate from side to side to ensure the cells are equally spread in the wells, or the cells will gather in the center of the wells. Inspect under a light microscope to make sure the cell is evenly spread in the media</li>
	<li>Put the plate in a conventional CO2 incubator at 37 &#176;C overnight or until the cells have adhered to the bottom of the wells.</li>
</ol>
3. Addition of Stimuli for Migration Effects
<ol>
	<li>Check if the wells are 90-95% confluent under a light microscope.</li>
	<li>Prepare 10 &#181;g/mL of mitomycin C in an appropriate amount of the medium. 
	Note: In this study, Mitomycin C is kept at 4 &#176;C in a stock solution at 1 mg/mL. Mitomycin C in solution can be stored up to one week in the dark at 2-8 &#176;C.</li>
	<li>Remove the spent media with a Pasteur pipette from each well.</li>
	<li>Add 250 &#181;L of 5 &#181;g/mL of mitomycin C to each well.</li>
	<li>Incubate the plate for 2 h at 37 &#176;C and 5% CO2.</li>
	<li>Prepare stimuli of peptides by diluting them in an appropriate amount of the medium. 
	Note: In this study, both the human and bovine Lactoferrin and Lactoferricin were tested at 25 &#181;g/mL in a volume of 250 &#181;L</li>
	<li>Remove the medium with mitomycin C with a Pasteur pipette.</li>
	<li>Wash the wells 1x with 250 &#181;L of 1x PBS (room temperature) and remove the wash with a Pasteur pipette.</li>
	<li>Add 250 &#181;L of 1x PBS and manually scratch the wells vertically with a 200 &#181;L pipette tip. Use a new pipette tip for each well.</li>
	<li>Remove the PBS and add 250 &#181;L peptide stimuli.</li>
	<li>Mount the plate in optical camera system by adjusting the screws on both sides of the plate. Leave the lid on the microtiter plate. 
	Note: The camera can operate with the lid open if necessary.</li>
</ol>
4. Set Up the Program for the Optical Camera on the Computer
<ol>
	<li>Click Acquire in the taskbar on the left.</li>
	<li>Choose the function Wound healing in the taskbar on the left.</li>
	<li>Choose the plate type by clicking below the Plate illustration.</li>
	<li>Unselect wells, not in use, by marking them and click Enable at the upper left side on the screen. 
	Note: All wells are selected by default.</li>
	<li>Adjust the focus of the lens on the wells to fit the position of the cells by right-clicking on one of the wells. 
	Note: Autofocus can also be adjusted manually by moving the bar to the right of the plate illustration.</li>
	<li>Set the time period for images under Picture Interval (e.g. (00:30:00)).</li>
	<li>Set the number of repetition to fit the desired period (e.g. 97 repetitions).</li>
	<li>Start the run by clicking Start button in the lower right corner.</li>
</ol>

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<li>Räsänen, K., Vaheri, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TGF-beta1+causes+epithelial-mesenchymal+transition+in+HaCaT+derivatives%2C+but+induces+expression+of+COX-2+and+migration+only+in+benign%2C+not+in+malignant+keratinocytes%5BTitle%5D)+AND+%22Journal+of+Dermatological+Science%22%5BJournal%5D)">TGF-beta1 causes epithelial-mesenchymal transition in HaCaT derivatives, but induces expression of COX-2 and migration only in benign, not in malignant keratinocytes.</a> Journal of Dermatological Science. 58 (2), 97-104 (2010).</li>
<li>Tochio, T., Tanaka, H., Nakata, S., Hosoya, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fructose-1%2C6-bisphosphate+aldolase+A+is+involved+in+HaCaT+cell+migration+by+inducing+lamellipodia+formation%5BTitle%5D)+AND+%22Journal+of+Dermatological+Science%22%5BJournal%5D)">Fructose-1,6-bisphosphate aldolase A is involved in HaCaT cell migration by inducing lamellipodia formation.</a> Journal of Dermatological Science. 58 (2), 123-129 (2010).</li>
<li>Tomasz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitomycin+C%3A+small%2C+fast+and+deadly+%28but+very+selective%29%5BTitle%5D)+AND+%22Chemistry+and+Biology%22%5BJournal%5D)">Mitomycin C: small, fast and deadly (but very selective).</a> Chemistry and Biology. 2 (9), 575-579 (1995).</li>
<li>Stamm, A., Reimers, K., Strauß, S., Vogt, P., Scheper, T., Pepelanova, I. <a target="_blank" href="https://www.degruyter.com/view/j/biomat.2016.17.issue-1-2/bnm-2016-0002/bnm-2016-0002.xml">In vitro wound healing assays - state of the art.</a> BioNanoMaterials. 17 (1-2), 79-87 (2016).</li>
<li>Justus, C. R., Leffler, N., Ruiz-Echevarria, M., Yang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+Cell+Migration+and+Invasion+Assays%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">In vitro Cell Migration and Invasion Assays.</a> Journal of Visualized Experiments. (88), 1-8 (2014).</li>
<li>Jonkman, J. E. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+introduction+to+the+wound+healing+assay+using+live-cell+microscopy%5BTitle%5D)+AND+%22Cell+Adhesion+%26+Migration%22%5BJournal%5D)">An introduction to the wound healing assay using live-cell microscopy.</a> Cell Adhesion & Migration. 8 (5), 440-451 (2016).</li>
<li>Hong, J., Kandasamy, K., Marimuthu, M., Choi, C. S., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrical+cell-substrate+impedance+sensing+as+a+non-invasive+tool+for+cancer+cell+study%5BTitle%5D)+AND+%22The+Analyst%22%5BJournal%5D)">Electrical cell-substrate impedance sensing as a non-invasive tool for cancer cell study.</a> The Analyst. 136 (2), 237-245 (2011).</li>
<li>Fredborg, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time+optical+antimicrobial+susceptibility+testing%5BTitle%5D)+AND+%22Journal+of+Clinical+Microbiology%22%5BJournal%5D)">Real-time optical antimicrobial susceptibility testing.</a> Journal of Clinical Microbiology. 51 (7), 2047-2053 (2013).</li>
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The authors have nothing to disclose.

The importance of migration in studies of development, wound healing, immunology, and cancer has led to several different methods to study migration. Migration can either be an expression of the expansion or the closure of the gap by the cells. Most often closure of the scratch is tested, as it is easier to grow cells in a monolayer and then make a scratch than growing the cells in a specific form8. Our study demonstrates an optimized method for manual scratching, as the technique is easy to maste...

Migration is an important process that influences several physiological aspects. In wound healing, migration facilitates re-epithelization of the skin. In non-healing wounds, such as chronic wounds, opportunistic bacteria cause infection of the wound, and open wounds are ideal for bacteria growth and formation of biofilm1,2. Biofilm is one of the causes of the development of resistant bacteria, which is one of the biggest threats to our modern society3. In wound healing, the immune cells cannot penetrate the biofilm. In cancer, migration of the cancer cells is a pivotal step of metastasis, which is the primary cause of death for patients with solid tumors4,5. Therefore, it is crucial to be able to study migration.
The scratch assay is often used to test cell migration because it is cheap and easy to perform on adherent cell lines, such as fibroblast, endothelial, and epithelial cell lines6,7. Today, several methods exist for performing scratches8, and different materials have been reported to be used, including toothpicks9, cell scrapers10, lasers11, and electric currents12. All of the methods have different pros and cons, and the method should be decided upon according to the cell type, budget, and analysis tool. As an example, if the used cell type requires coating, manual pressure removal, e.g. with a toothpick or pipette tip, will influence migration within the area, a different method should be used. In this study, we will focus on manual scraping.
A downside for manual scraping is the variation of the size, shapes of the scratches, and accumulation of cell on the edges of the scratch. Many protocols exist, and a highly cited paper advises increased speed and/or thorough washing after scratching13. Additionally, several methods have been used to minimize proliferation, since the proliferation of the cells cannot be distinguished from migration and therefore may give false-positive results of migration. Some reported methods are serum starvation preceding the scratch14 and decreasing the amount of serum15. However, neither of these methods can ensure that there is no proliferation. Therefore, we report a pre-treatment of the cells with mitomycin C to ensure true results of migration. Mitomycin C is an antibiotic that inhibits DNA synthesis by forming a covalent bond with the DNA, which prevents the DNA from separating16. By pre-treating with mitomycin C and washing with PBS before scratching, the detected closure of the gap demonstrates the true migration of the cells (Figure 1).
A characteristic of all the methods is the need for a system to analyze the process of migration17. A common and an inexpensive method to analyze the progress is to use a light-field microscope to take images of the scratch at pre-determined time points, followed by an analysis of the closure of the gap in an image software18,19. This method is time-consuming, unreliable with regards to taking pictures at the same spot and focus, and the movement from incubator to camera forces cycles of temperature changes while pictures are taken. Other methods have been developed to detect migration by measuring the current flow. The current changes, as cells cover more space on the plate, which is read as an increase in the impedance20. By measuring impedance, the environment of the cells is not affected, and the method is therefore considered non-invasive. This method relies on specialized plates with gold electrodes, which are expensive, but they enable detection of many processes, such as cellular adherence, proliferation, migration, and cell death. A big disadvantage of this method is that the impedance measurement does not directly indicate migration, as factors such as temperature have a great impact on the impedance. Changes in the impedance can be caused by several factors that are not reviewed by the software, making the analysis of the data more difficult. Therefore, the lack of visualization requires a conventional light microscope to verify migration.
To overcome many of the downsides of the available techniques, we use a real-time automated optical camera. The optical camera is a detection system with a high-throughput microscope in a small platform with a lens, illumination unit, and a digital camera. It has a built-in software, which can test migration and proliferation among other things. To detect migration, two algorithms are used to analyze the growth and the migration kinetics of cells, which are called the proliferation and the wound healing analysis, respectively. The proliferation analysis is based on a two-step image segmentation algorithm that applies texture analysis to detect the difference between cell-covered areas (shown in yellow) and empty background areas by advanced histogram analysis. The wound healing analysis is based on a three-step algorithm that detects the cell monolayer, locates the wound gap, and determines the gap width. These steps are conducted at user-determined time-points, and the data is presented as covered area, gap width, and gap area. One of the great advantages of this machine is that it enables imaging of adherent cells through liquid because of the angle of the camera21. The camera lenstakes tilted images that are projected to a z-stack of a single z-plane generate to ensure that the pictures are in-focus (Figure 2). z-stacks are generated by merging the series of pictures (e.g. 40) taken perpendicular to the wound edge, and across the entire wound. The z-stacks enables capturing of the depth of the cell layer as well as an in-focus plane across the wound. Furthermore, the series of images can be put together to a video collection of the images, with the click of a button.
We demonstrate an optimized protocol for detection of migration in the keratinocyte cell line HaCaT. We tested Lactoferrin and its N-terminal peptide, Lactoferricin, from humans and cows, to analyze their effect on migration as these molecules have been demonstrated to have numerous applications as antimicrobial and immune modulating molecules22,23. The four compounds were compared to untreated HaCaT cells and epidermal growth factor (EGF) was used as a positive control.

<table><tbody><tr><td>oCelloScope</td><td>BioSense Solution ApS</td><td></td><td>Optical camera</td></tr><tr><td>UniExplorer software&amp;nbsp;</td><td>BioSense Solution ApS</td><td></td><td>(8.0.0.7144 (RL3))</td></tr><tr><td>HaCaT cell</td><td></td><td></td><td>Donated from Bispebjerg Hospital</td></tr><tr><td>DMEM (1x) + GlutaMAX</td><td>Gibco</td><td>31966-021</td><td>Medium for HaCaT</td></tr><tr><td>FBS</td><td>Gibco</td><td>10270</td><td>Fetal bovine serum</td></tr><tr><td>PBS</td><td>Gibco</td><td>D837</td><td>Without Mg+ and Ca2+</td></tr><tr><td>Pencillin-Streptomycin</td><td>Sigma</td><td>P0781</td><td>Antibiotics</td></tr><tr><td>Trypsin (10x)</td><td>Sigma</td><td>T3924</td><td></td></tr><tr><td>Mitomycin C</td><td>Tocris</td><td>3258</td><td>Inhibits proliferation</td></tr><tr><td>Microtiter plate</td><td>Greiner Bio-one</td><td>677 180</td><td></td></tr><tr><td>Incubator</td><td>Panasonic</td><td></td><td>37&amp;deg;C, 5% CO2</td></tr><tr><td>Hemocytometer</td><td>Assistent</td><td></td><td>T&amp;uuml;rk (0.1 mm)</td></tr><tr><td>Pipet boy</td><td>Accu-Jet pro</td><td></td><td></td></tr></tbody></table>

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.

HaCaT is a keratinocyte cell line, one of the most abundant cell types in the skin. When a wound occurs, the skin cells start to proliferate and migrate over to the wound bed to close the wound (Figure 3). Both processes are, therefore, of utmost importance for proper healing.
The optical camera can follow both processes in real-time. For migration, the system gives three data sets: cell covered are...

Watch this Scientific Journal Video about Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera at JoVE.com

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.

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 ...

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23.2K Views.  Roskilde University. Hier beschrijven we een procedure om te testen de cel migratie in vitro met een geautomatiseerde optische camera Microscoop. De kras assay is wijd verbeid gebruikt wanneer de sluiting van de kras gedurende een ingestelde periode wordt gevolgd. De optische Microscoop kunt betrouwbare en goedkope detectie van cel migratie.

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

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

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 ...

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

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast 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 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

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 ...

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 ...

Elucidating T-Cell Kinetics: Methods for Real-Time Migration Analysis

Real-Time In Vitro Migration Assay for Primary Murine CD8+ T Cells

The adaptive immune response is reliant on a T cell&#39;s ability to migrate through blood, lymph, and tissue&#160;in response to pathogens and foreign bodies. T cell migration is a complex process that requires the coordination of many signal inputs from the environment and local immune cells, including chemokines, chemokine receptors, and adhesion molecules. Furthermore, T cell motility is influenced by dynamic surrounding environmental cues, which can alter activation state, transcriptional landscape, adhesion molecule expression, and more. In vivo, the complexity of these seemingly intertwined factors makes it difficult to distinguish individual signals that contribute to T cell migration. This protocol provides a string of methods from T cell isolation to computer-aided analysis to assess T cell migration in real-time&#160;under highly specific environmental conditions.&#160;These conditions&#160;may help&#160;elucidate mechanisms that regulate migration, improving our&#160;understanding of T cell kinetics and providing&#160;strong mechanistic evidence that is difficult&#160;to attain through animal experiments. A deeper understanding of the&#160;molecular interactions that impact cell&#160;migration is important to develop improved therapeutics.

Author Spotlight: Understanding Disease Mechanisms Through Real-Time Analysis of T-Cell Migration

This protocol providesa method of primary murine T cell isolation and time-lapse microscopy of T cell migration under specific environmental conditions with quantitative analysis.

The adaptive immune response is reliant on a T cell&#39;s ability to migrate through blood, lymph, and tissue&#160;in response to pathogens and foreign ...

Immunology and Infection

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.

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

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