Name: scratch migration assay and dorsal skinfold chamber for in vitro and in vivo analysis of wound healing
Uploaded: 2019-09-26T13:00:00.000+00:00
Duration: 9 min 34 s
Description: Watch this Scientific Journal Video about Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing at JoVE.com

We thank Dr. Petra Weissgerber and the Transgene Unit of the SPF animal facility (project P2 of SFB 894) of the Medical Faculty and the animal facility at the Institute of Clinical and Experimental Surgery at the Medical Faculty of Saarland University, Homburg. We thank Dr. Andreas Beck for critical reading of the manuscript. This study was funded by the Deutsche Forschungsgemeinschaft (DFG) Sonderforschungsbereich (SFB) 894, project A3 to A.B. and V.F.).

All experimental procedures were approved and performed in accordance with the ethics regulations and the animal welfare committees of Saarland and Saarland University.
1 Primary cell culture and siRNA transfection
NOTE: In the described method, primary fibroblasts are used. These cells play a crucial role in wound healing and tissue remodeling11. In this experiment, the Cacnb3 gene, encoding the Cav&#946;3 subunit of high voltage-gated calcium channels12 was down-regulated, thereby showing its role in cell migration in vitro and skin wound repair in vivo4.
<ol>
	<li>Preparation of siRNA: Before reconstituting the siRNAs, briefly centrifuge the tubes to ensure that the content is at the bottom. Reconstitute the siRNAs in 100 &#181;L RNase-free buffer (100 mM potassium acetate, 30 mM HEPES, pH 7.5) provided by the manufacturer at a concentration of 20 &#181;M. This is a stock solution of siRNAs.</li>
	<li>Aliquot this stock solution at 10 &#181;L per tube (20 &#181;M concentration) and store at -20 &#176;C until use.</li>
	<li>Using an ultrafine permanent marker, mark a 6-well plate with a horizontal line at the bottom of each well in order to be able to always identify the same scratch region of interest and to follow its closure. 
	NOTE: 6-well culture plates were used in this assay because they are large enough, to provide enough space and flexibility to apply a consistent, reproducible and vertical scratch using a 200 &#181;L pipette tip across the cell monolayer. If a limited number of cells are available, an alternative and probably the cost-efficient way would be to use 12- or 24-well culture plates.</li>
	<li>Plate primary fibroblasts, isolated from the wild-type and &#946;3-deficient mice4, in a 6-well plate at a density of 5 x 105 cells/well in the presence of 2 mL Dulbecco&#8217;s modified Eagle&#39;s medium (DMEM) supplemented with 10% fetal bovine serum (FCS). 
	NOTE: 5 x 105 cells per well has been established for 6-well culture plate and for primary mouse fibroblasts. Tests may be needed if using 12- or 24-well cell culture plates or other cell types, which could be different in size. Cells should be handled in a sterile environment such as biological safety cabinets class II.</li>
	<li>Label the 6-well plate with the cell type, genotype, and the date.</li>
	<li>Move the 6-well plate into the cell culture incubator and maintain cells at 37 &#176;C and 5% CO2 for 24 h.</li>
	<li>The next day, take the plate out of the incubator, aspirate the cell culture medium out of the well, discard it and replace it with 2.25 mL fresh culture medium by adding it carefully against the wall of the well.</li>
	<li>In order to transfect fibroblasts with siRNAs, use a lipid-based transfection reagent as recommended by the manufacturer.</li>
	<li>For each transfection, label two microcentrifuge tubes. In the first one, add 9 &#181;L of the transfection reagent and dilute it with 150 &#181;L reduced serum medium. In the second tube, add 1.5 &#181;L siRNA (Cacnb3 siRNA-1, Cacnb3 siRNA-2 or scrambled siRNA as a negative control) and dilute it with 150 &#181;L reduced serum medium.</li>
	<li>Add the diluted siRNA into the tube containing diluted transfection reagent and vortex for 2 s. Incubate the mixture for 5 min at 21 &#176;C.</li>
	<li>Label wells with either Cacnb3 siRNA-1, Cacnb3 siRNA-2 or scrambled siRNA. Add 250 &#181;L of the siRNA-transfection reagent mixture dropwise to the cells.</li>
	<li>Place the 6-well culture plate back into the incubator and keep cells at 37 &#176;C and 5% CO2 for 72 h.</li>
	<li>In order to check the efficiency of Cacnb3 gene silencing, collect transfected cells and perform immunoblot analysis as described previously4.</li>
</ol>
2. In vitro wound healing assay (scratch migration assay)
<ol>
	<li>Take the cell culture plate out of the incubator and examine the cells under the microscope using the 10x objective. Start with the scratch assay only when they have reached 100% confluency. 
	NOTE: For the accuracy and reproducibility, 100% confluency is a mandatory factor for starting the scratch migration assay. Therefore, it is important to seed the same number of cells into the culture wells, to examine each well for confluency and to apply the scratch at the same time point (day 0 confluency). Waiting for longer after the cells reach 100% confluency can evoke different responses.</li>
	<li>Once the cell reaches 100% confluency, aspirate the culture medium out of the well and discard it.</li>
	<li>Use a pipette tip (200 &#181;L) to manually create a scratch vertical to the horizontal line marked at the bottom of the well, across the confluent cell monolayer in the middle of the well.</li>
	<li>Rinse each well twice with 2 mL phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.4) to remove the released factors from damaged cells, loose cells, and debris from the scratched area. Add 2 mL of PBS carefully against the wall of the well to avoid detaching cells from the cell culture well.</li>
	<li>Add 2 mL of cell culture medium containing either 10% serum or 1% serum carefully to each well. 
	NOTE: It is recommended to perform the scratch assay under 10% serum and under 1% serum to confirm that the observed effect is caused by the cell proliferation and migration or by cell migration only.</li>
	<li>Move the plate to the microscope stage and capture bright field images of the cell-free area (two areas per well) immediately after scratching (t=0h) at a 10x magnification using a light microscope. To image always the same region of the scratch, use the horizontal line, which was prepared in step (1.3), and take one image above this line and one image below this line. Save images as TIFF or JPEG.</li>
	<li>Because the microscope stage does not maintain the cell growth condition, move the plate back to the cell culture incubator and keep the cells at 37 &#176;C and 5% CO2.</li>
	<li>After 6, 10 and 30 h, move the plate to the microscope stage again and capture images the same way as described in step 2.6. 
	NOTE: These time points have been established for the described procedure and for primary fibroblasts. During the first pilot experiment, more time points were tested to see how fast fibroblasts repopulate the gap. Although 0, 6, 10 and 30 h are reasonable starting time points, the investigators should optimize and establish the appropriate time points for each application and for each cell type. The more accurate alternative, if available, would be to use time-lapse microscopy.</li>
	<li>Using ImageJ13, quantify the initial cell-free area (100%) and the remaining area after 6, 10 and 30 h (Figure 1). The percentage of scratch area repopulated by migrating cells is then calculated relative to the initial scratch area.</li>
</ol>
3. Analysis of the scratch area
<ol>
	<li>Open ImageJ software13.</li>
	<li>Upload the first image as JPEG (e.g., 24-bit RGB images 1360x1024) by dropping the image into the ImageJ menu bar.</li>
	<li>Select the Freehand selections button and mark the cell-free area</li>
	<li>Click on Analyze and select Measure. A window with the results will appear containing the area value.</li>
	<li>Transfer this value into an analysis spreadsheet.</li>
	<li>Repeat steps 3.2-3.5 for each image from time point 0 h and then start again for the next time points 6, 10 and 30 h.</li>
	<li>Calculate the percentage of scratch area repopulated by migrating cells after 6, 10 and 30 h for each scratch using the following equation: 
	<img alt="Equation 1" src="/files/ftp_upload/59608/59608eq1.jpg" /> 
	a = cell free area of the initial scratch, b = cell free area after 6 h</li>
	<li>Calculate the mean and the standard error of the mean (S.E.M.) for the percentage of scratch area repopulated by migrating cells after 6 h. Show data as a column bar graph or a scatter plot.</li>
</ol>
4. In vivo skin wound healing assay
NOTE: C57BL/6 wild-type males (8-12 weeks old with 22-26 g body weight) and Cav&#946;3-deficient mice as a control are used for this study.
<ol>
	<li>One day before starting the experiment, autoclave all the surgical instruments, screws, nuts and titanium frames to be used for the skinfold chamber preparation. 
	NOTE: The titanium frame is composed of two symmetrical complementary halves and it has a circular observation window where the wound will be applied and followed by microscopy (see Figure 2a).</li>
	<li>Anesthetize a wild-type or &#946;3-deficient mouse (22-26 g body weight) by intraperitoneal (i.p.) injection of 0.1 mL saline/10 g body weight containing a mixture of ketamine (75 mg/kg body weight) and xylazine (25 mg/kg body weight). Check the depth of anesthesia by the lack of response to a toe pinch. 
	NOTE: This injection gives around 30 min surgical anesthesia and the depth of anesthesia must be controlled through the surgical procedure, by checking the reflexes of the mouse.</li>
	<li>To avoid dryness or damage of the eyes, apply ophthalmic ointment to both eyes and repeat the application if necessary.</li>
	<li>Carefully shave the mouse dorsum, using an electric shaver followed by the application of a depilation cream to the shaved area to remove any remaining hair. Take care not to injure the mouse skin. Leave the depilation cream for around 10 min to completely remove all hair.</li>
	<li>Prepare the titanium chamber by taking one part of the symmetrical titanium chamber frame and fix the connecting screws with nuts on one side. These nuts will serve as a spacer to keep 400-500 &#181;m between the two symmetrical parts of the chamber to avoid compression of blood vessels in the skin.</li>
	<li>Remove the cream from the back of the mouse and clean the hair-free region with warm (35-37 &#176;C) tap water.</li>
	<li>Make sure that the place to perform surgery is clean, warm (37 &#176;C) and humidified.</li>
	<li>Disinfect the hair-free area of the mouse with skin disinfectant. Take a fold of the back skin of the mouse in front of a light source and position the middle line of the double layer of the skin where the titanium chamber will be implanted. After that, fix the skinfold with a polypropylene suture cranially and caudally and tighten the other side of the suture on a metal rack to lift the mouse folded skin. Adjust the height of the rack to allow the mouse to sit comfortably.</li>
	<li>Implant the titanium chamber into the fold of the back skin of the mouse in a way to sandwich the folded dorsal skin layer between the two symmetrical halves of the titanium frame. Attach the first half of the titanium frame by polypropylene sutures on its superior edge to the back of the dorsal skinfold. 
	NOTE: On the titanium frame, there are 8 holes on the superior edge (Figure 2a) and the folded skin should be well fixed by polypropylene sutures on each of the eight holes.</li>
	<li>Before moving to the next step, check the reflexes of the mouse to make sure that the depth of anesthesia is maintained.</li>
	<li>At the base of the skinfold, pass the two connecting screws, attached to the first half of the titanium chamber, to penetrate the skinfold from back to the front side. Make small incisions on the skin (using fine scissors) to help smooth penetration of the connecting screws.</li>
	<li>Detach the mouse from the rack and place it on a lateral position. Put the second complementary half of the titanium chamber on top of the 3 connecting screws (see Figure 2a) and apply slight pressure with fingers in order to pass these screws through the second half of the titanium frame. Then, fix both symmetrical parts with stainless steel nuts.</li>
	<li>Pay careful attention to the tightness of the screws at this step, since it might detach, if it is too loose. In contrast, if it is too tight, it will squeeze the skinfold, reduce blood flow and can lead to tissue impairment and necrosis. 
	NOTE: Nuts prepared in step 4.5 serve as a spacer to keep a distance of 400-500 &#181;m between the two symmetrical halves of the titanium chamber. The nuts should be tightened until a slight resistance is felt.</li>
	<li>Cut the remaining part of the screws using pliers. 
	NOTE: It is necessary at this step to use laboratory safety glasses for eye protection in case the screw comes off the wrong way.</li>
	<li>Mark the wound area by a standardized biopsy punch (2 mm in diameter), in the center of the skin within the observation window (see Figure 2a) of the skinfold chamber in order to ensure reproducible wound sizes.</li>
	<li>By using fine forceps and scissors, create a circular wound within the marked area by removing the complete skin with epidermis and dermis. The final wound area will be around 3.5-4.5 mm2, see Figure 2b. Clean the wound with 0.5 mL sterile saline (0.9 % NaCl, 37 &#176;C).</li>
	<li>Cover the wound with a glass coverslip and fix this glass coverslip with a snap ring using the snap ring plier on the titanium chamber.</li>
	<li>Immediately after finishing the surgical procedure, place the mouse on the imaging stage of a stereomicroscope equipped with a camera and take images (day 0) under illumination. Use the 40X magnification and save the images for future off-line analysis. 
	NOTE: The investigator should examine the images immediately after capture to ensure that quality is sufficient for future off-line analyses. The preparation of the skinfold chamber and performance of the skin wound takes around 30 minutes.</li>
	<li>Keep the mouse at a warm place during recovery from anesthesia for at least 2 h. Thereafter, transfer mice in individual cages back to the animal facility (12 h light/dark cycle) and make sure that mice have access to food and water.</li>
	<li>Three days post-wounding place the mouse in a mouse-restrainer and fix the restrainer on top of the imaging stage.</li>
	<li>Place the stage under a stereomicroscope equipped with a camera. Take images under illumination with 40x magnification, record all pictures and save them for future off-line analyses</li>
	<li>Repeat steps 4.20 and 4.21 again at day 6, 10 and day 14 post-wounding.</li>
	<li>Use the wound images, for off-line analysis in ImageJ13. The wound area at day 0 is considered as 100 % and the wound closure over time is plotted relative to the initial wound area. Representative results are shown in Figure 2c,d.</li>
	<li>Calculate the percentage (%) of the wound area at each time point using the following equation: 
	<img alt="Equation 2" src="/files/ftp_upload/59608/59608eq2.jpg" /> 
	x: time point (day 0, 3, 10 or 14), a: wound area at day 0, b: wound area at time point x</li>
</ol>

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The authors have nothing to disclose.

In this manuscript, we describe an in vitro and in vivo wound healing assay and correlate the results obtained. For the in vitro assay, we used primary mouse fibroblasts4,14,15 which play an important role in wound healing and tissue remodeling11. Other adherent cell types growing as monolayers (e.g., epithelial cells, endothelial cells, keratinocytes) can be used as well. Plating the same number of viabl...

Skin wound healing starts immediately after the skin injury in order to restore the skin&#39;s integrity and to protect the organism from infections. The wound healing process goes through four overlapping phases; coagulation, inflammation, new tissue formation, and tissue remodeling1. Cell migration is crucial during these phases. Inflammatory cells, immune cells, keratinocytes, endothelial cells, and fibroblasts are activated at different time points and invade the wound area2. Methods to investigate wound healing in vitro and in vivo are of great interest not only to understand the underlying mechanisms but also to test new drugs and to develop new strategies aiming to ameliorate and accelerate skin wound healing.
To monitor and analyze cell migration, the scratch migration assay can be used. It is often referred to as in vitro wound healing assay. This method requires a cell culture facility3. It is a simple procedure, there is no need of high-end equipment and the assay can be performed in most cell biology laboratories. In this assay, a cell-free area is created by the mechanical disruption of a confluent cell monolayer, preferably epithelial- or endothelial-like cells or fibroblasts. Cells on the edge of the scratch will migrate in order to repopulate the created gap. Quantification of the decreasing cell-free area over time resembles the migration rate and indicates the time, which the cells need to close the gap. For this purpose, investigators can use either acutely isolated cells from WT mice or mice lacking a gene of interest4, or immortalized cells available from reliable cell repositories. The scratch assay allows studying the influence of pharmacologically active compounds or the effect of transfected cDNAs or siRNAs on cell migration.
In vivo, wound healing is a complex physiological process, requiring different cell types including keratinocytes, inflammatory cells, fibroblasts, immune cells and endothelial cells in order to restore the skin&#8217;s physical integrity as fast as possible1. Different methods to study in vivo wound healing have been developed and used in the past5,6,7,8. The dorsal skinfold chamber described in this article was previously used for wound healing assays9. It is used as a modified dorsal skinfold chamber preparation for mice. The modified skinfold chamber model has several advantages. 1) It minimizes skin contraction, which prevents observing the wound healing process and might influence wound repair in mice. 2) This chamber makes use of covering the wound with a glass coverslip, reducing tissue infections and desiccation, which could delay the healing process. 3) Blood flow and vascularization can be directly monitored. 4) It allows repetitive topical application of pharmacologically active compounds and reagents in order to treat the wound and accelerate healing9,10.
We identified the &#946;3 subunit of high voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence skin wound healing using two independent protocols, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. For the in vitro assay, we used primary fibroblasts, these cells do express the Cacnb3 gene encoding the Cav&#946;3 protein but lack depolarization-induced Ca2+ influx or voltage-dependent Ca2+ currents. We described a novel function of Cav&#946;3 in these fibroblasts: Cav&#946;3 binds to the inositol 1,4,5-trisphosphate receptor (IP3R) and constraints calcium release from the endoplasmic reticulum. Deletion of the Cacnb3 gene in mice leads to increased sensitivity of the IP3R for IP3, enhanced cell migration and increased skin wound repair4.

<table><tbody><tr><td>0.9 % NaCl</td><td></td><td></td><td></td></tr><tr><td>1 ml syringes</td><td>BD Plastipak</td><td>303172</td><td></td></tr><tr><td>6 well plate</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>Biopsy punch</td><td>Kai Industries</td><td>48201</td><td>2 mm</td></tr><tr><td>Cacnb3 Mouse siRNA Oligo Duplex (Locus ID 12297)</td><td>Origene</td><td>SR415626</td><td></td></tr><tr><td>Depilation cream</td><td></td><td></td><td>any depilation cream</td></tr><tr><td>Dexpanthenol 5% (BEPANTHEN)</td><td>Bayer</td><td>3400935940179.00</td><td>(BEPANTHEN)</td></tr><tr><td>Dihydroxylidinothiazine hydrochloride (Xylazine)</td><td>Bayer Health Care</td><td></td><td>Rompun 2%</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Gibco by life technologies</td><td>41966-029</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco by life technologies</td><td>10270-106</td><td></td></tr><tr><td>Hexagon full nut</td><td></td><td></td><td></td></tr><tr><td>Ketamine hydrochloride</td><td>Zoetis</td><td></td><td>KETASET</td></tr><tr><td>Light microscope</td><td>Keyence, Osaka, Japan</td><td>BZ-8000</td><td>Similar microscopes might be used</td></tr><tr><td>Lipofectamine RNAiMAX Transfection Reagent</td><td>Thermo Fisher Scientific</td><td>13778075</td><td></td></tr><tr><td>Micro-forceps</td><td></td><td></td><td></td></tr><tr><td>Micro-Scissors</td><td></td><td></td><td></td></tr><tr><td>Mouse restrainer</td><td>Home-made</td><td></td><td></td></tr><tr><td>Normal scissors</td><td></td><td></td><td></td></tr><tr><td>Objective</td><td>Nikon</td><td>plan apo 10x/0.45</td><td></td></tr><tr><td>Opti-MEM</td><td>Gibco by life technologies</td><td>51985-026</td><td></td></tr><tr><td>Polypropylene sutures</td><td></td><td></td><td></td></tr><tr><td>Screwdriver</td><td></td><td></td><td></td></tr><tr><td>Skin disinfectant (octeniderm)</td><td>Sch&amp;uuml;lke &amp;amp; Mayr GmbH</td><td>118212</td><td></td></tr><tr><td>Slotted cheese head screw</td><td></td><td></td><td></td></tr><tr><td>Snap ring</td><td></td><td></td><td></td></tr><tr><td>Snap ring plier</td><td></td><td></td><td></td></tr><tr><td>Surgical microscope with camera</td><td>Leica</td><td>Leica M651</td><td></td></tr><tr><td>Titanium frames for the skinfold chamber</td><td>IROLA</td><td>160001</td><td>Halteblech M</td></tr><tr><td>Wire piler</td><td></td><td></td><td></td></tr></tbody></table>

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.

The scratch assay was performed on a confluent cell monolayer of wild-type and &#946;3-deficient MEFs (Figure 1c). After performing the &#34;scratch&#34;&#160;using a 200 &#956;L pipette tip, cells from both genotypes migrate into the scratch area and close the gap. Images were taken after 6, 10 and 30 h (Figure 1a). Cell migration was quantified as the percentage (%) of scratch area repopulated by migrating cells 6 hours after ...

Watch this Scientific Journal Video about Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing at JoVE.com

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

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 ...

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Research

JoVE Journal

Medicine

13.1K Views.  Saarland University. 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.

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

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 ...

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

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 ...

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine models provide valuable mechanistic insight into these processes; however, no single model fully addresses all aspects of the wound healing response. Instead, it is ideal to use multiple models to address the different aspects of wound healing. Here, two different methods that address diverse aspects of the wound healing response are described. In the first model, polyvinyl alcohol sponges are subcutaneously implanted along the mouse dorsum. Following sponge retrieval, cells can be isolated by mechanical disruption, and fluids can be extracted by centrifugation, thus allowing for a detailed characterization of cellular and cytokine responses in the acute wound environment. A limitation of this model is the inability to assess the rate of wound closure. For this, a tail skin excision model is utilized. In this model, a 10 mm x 3 mm rectangular piece of tail skin is excised along the dorsal surface, near the base of the tail. This model can be easily photographed for planimetric analysis to determine healing rates and can be excised for histological analysis. Both described methods can be utilized in genetically altered mouse strains, or in conjunction with models of comorbid conditions, such as diabetes, aging, or secondary infection, in order to elucidate wound healing mechanisms.

Here, two murine wound healing models are described, one designed to assess cellular and cytokine wound healing responses and the other to quantify the rate of wound closure. These methods can be used with complex disease models such as diabetes to determine mechanisms of various aspects of poor wound healing.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine ...

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

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 ...

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

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Chapters in this video

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

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

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

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

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

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

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

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

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

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