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.

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			<td>0.9 % NaCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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			<td>1 ml syringes</td>
			<td>BD Plastipak</td>
			<td>303172</td>
			<td></td>
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			<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>
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			<td>Depilation cream</td>
			<td></td>
			<td></td>
			<td>any depilation cream</td>
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			<td>Dexpanthenol 5% (BEPANTHEN)</td>
			<td>Bayer</td>
			<td>3400935940179.00</td>
			<td>(BEPANTHEN)</td>
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		<tr>
			<td>Dihydroxylidinothiazine hydrochloride (Xylazine)</td>
			<td>Bayer Health Care</td>
			<td></td>
			<td>Rompun 2%</td>
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			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco by life technologies</td>
			<td>41966-029</td>
			<td></td>
		</tr>
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			<td>Fetal bovine serum</td>
			<td>Gibco by life technologies</td>
			<td>10270-106</td>
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			<td>Hexagon full nut</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Ketamine hydrochloride</td>
			<td>Zoetis</td>
			<td></td>
			<td>KETASET</td>
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			<td>Light microscope</td>
			<td>Keyence, Osaka, Japan</td>
			<td>BZ-8000</td>
			<td>Similar microscopes might be used</td>
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			<td>Lipofectamine RNAiMAX Transfection Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>13778075</td>
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			<td>Micro-forceps</td>
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			<td>Micro-Scissors</td>
			<td></td>
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			<td>Mouse restrainer</td>
			<td>Home-made</td>
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			<td>Normal scissors</td>
			<td></td>
			<td></td>
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			<td>Objective</td>
			<td>Nikon</td>
			<td>plan apo 10x/0.45</td>
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			<td>Opti-MEM</td>
			<td>Gibco by life technologies</td>
			<td>51985-026</td>
			<td></td>
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			<td>Polypropylene sutures</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Screwdriver</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>Skin disinfectant (octeniderm)</td>
			<td>Sch&#252;lke &#38; 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>
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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 ...

scratch-migration-assay-dorsal-skinfold-chamber-for-vitro-vivo

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JoVE Journal

Medicine

13.0K 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

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

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 ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...