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

1. General cell culture
<ol>
	<li>Culture cardiac fibroblasts in Dulbecco&#8217;s Modified Eagles Medium (DMEM) containing 1 g/L glucose, sodium pyruvate, L-glutamine, and supplemented with 14.2 mM NaHCO3, 14.9 mM HEPES, 15% heat-inactivated fetal bovine serum (FBS), 2% L-glutamine, and 0.02% antimicrobial reagent (see Table of Materials) and maintained in CO2 incubator at 37 &#176;C.</li>
	<li>Culture cardiac fibroblasts until 90-95% confluency is reached at passage 0 (P0). At th...

<ol>
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(17), 524-532 (2005).</li><li>Valster, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+and+invasion+assays.">Cell migration and invasion assays.</a> Methods. 37, 208-215 (2005).</li><li>Pijuan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cell+migration,+invasion,+and+adhesion+assays:+From+cell+imaging+to+data+analysis.">In vitro cell migration, invasion, and adhesion assays: From cell imaging to data analysis.</a> Frontiers in Cell and Developmental Biology. 7, 1-16 (2019).</li><li>Liang, C. C., Park, A. Y., Guan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+scratch+assay:+a+convenient+and+inexpensive+method+for+analysis+of+cell+migration+in+vitro.">In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro.</a> Nature Protocols. 2 (2), 329-333 (2007).</li><li>Hulkower, K. I., Herber, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+and+invasion+assays+as+tools+for+drug+discovery.">Cell migration and invasion assays as tools for drug discovery.</a> Pharmaceutics. 3 (1), 107-124 (2011).</li><li>Walter, M. N. M., Wright, K. T., Fuller, H. R., MacNeil, S., Johnson, W. E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell-conditioned+medium+accelerates+skin+wound+healing:+An+in+vitro+study+of+fibroblast+and+keratinocyte+scratch+assays.">Mesenchymal stem cell-conditioned medium accelerates skin wound healing: An in vitro study of fibroblast and keratinocyte scratch assays.</a> Experimental Cell Research. 316 (7), 1271-1281 (2010).</li><li>Chaudhary, A., Bag, S., Barui, A., Banerjee, P., Chatterjee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Honey+dilution+impact+on+in+vitro+wound+healing:+Normoxic+and+hypoxic+condition.">Honey dilution impact on in vitro wound healing: Normoxic and hypoxic condition.</a> Wound Repair and Regeneration. 23 (3), 412-422 (2015).</li><li>Lipton, A., Klinger, I., Paul, D., Holleyt, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+of+Mouse+3T3+Fibroblasts+in+Response+to+a+Serum+Factor.">Migration of Mouse 3T3 Fibroblasts in Response to a Serum Factor.</a> Proceedings of the National Academy of Sciences of the United States of America. 68 (11), (1971).</li><li>Ascione, F., Guarino, A. M., Calabr&#242;, V., Guido, S., Caserta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+to+quantify+the+wound+closure+dynamic.">A novel approach to quantify the wound closure dynamic.</a> Experimental Cell Research. (352), 175-183 (2017).</li><li>Burr, S. D., Harmon, M. B., S, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Impact+of+Diabetic+Conditions+and+AGE/RAGE+Signaling+on+Cardiac+Fibroblast+Migration.">The Impact of Diabetic Conditions and AGE/RAGE Signaling on Cardiac Fibroblast Migration.</a> Frontiers in Cell and Developmental Biology. 8, (2020).</li><li>Chang, S. S., Guo, W. H., Kim, Y., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidance+of+Cell+Migration+by+Substrate+Dimension.">Guidance of Cell Migration by Substrate Dimension.</a> Biophysical Journal. 104, 313-321 (2013).</li><li>Nguyen-Ngoc, K. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ECM+microenvironment+regulates+collective+migration+and+local+dissemination+in+normal+and+malignant+mammary+epithelium.">ECM microenvironment regulates collective migration and local dissemination in normal and malignant mammary epithelium.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (39), 2595-2604 (2012).</li></ol>

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

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

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

a cost effective and adaptable scratch migration assay

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

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

Watch this Scientific Journal Video about A Cost Effective and Adaptable Scratch Migration Assay at JoVE.com

A Cost Effective and Adaptable Scratch Migration Assay

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

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

This protocol provides a new method for an old technique, and it will provide a new approach for an large number of basic science researchers. The main advantages of this technique are it's cost effective nature as well as its adaptability to be used by a wider scientific audience. To prepare a migration plate, use a light colored permanent marker to draw a line down the center of the back of each well of a 48 well plate and to draw three hash marks to divide each well into three separate areas of interest.Then plate 1.5 to two times 10 to the fourth single passage cardiac fibroblasts into each well and culture the cells under normal culture conditions for 24 to 48 hours until they reach 90 to 95%confluency. When the cells are ready, remove the supernatant from each well and use a sterile P200 pipette tip to make a one pass scratch along the drawn lines. Rinse the wells with low serum medium to remove any unattached cardiac fibroblasts and add 500 microliters of low serum medium to each well.If pharmacological agents are being used, add the agents to the appropriate wells. Next, use an inverted microscope with a 20x objective to capture two zero hour images per well using the markings to position each well to allow imaging of the top half of the scratched line. Then move the plate to position the bottom half of the scratched line into view to collect the second image and place the plate in the cell culture incubator for 24 hours.At the end of the incubation, wash each well with non-sterile PVS and add 500 microliters of 4%paraformaldehyde to each well in a fume hood. After 10 minutes at room temperature, wash each well three times in fresh PBS for five minutes per wash. After the last wash, permeabilize the cells with 300 microliters of permeabilizing solution per well and gentle rocking for 30 minutes at room temperature.At the end of the incubation, replace the permeabilizing solution with 1%Kumasi brilliant blue stain for a 10 minute incubation with gentle rocking at room temperature. Followed by three, five minute washes in PBS with rocking. After the last wash, add 300 microliters of PBS to each well and carefully position the plate in the same position as for the zero hour image before capturing two 24 hour images per well as demonstrated.At the end of the experiment, open the images and an appropriate imaging analysis software program and create a new layer on the zero hour image. Double click to change the name of the new layer line layer and select the brush tool. Change the pixel size to 10 and the color to red and draw two separate lines to outline the scratch.The lines should not touch any cells. Open the 24 hour image in the imaging software and select the move tool. Holding down the control button, click both the lines and the background layers, click in the center of the zero hour image and drag both layers to the middle of the 24 hour image.In the 24 hour image, click the zero hour background layer and change the opacity to 50%Holding the control button, click both the zero hour background and line layers and click edit and free transform to free transform the layers Using free transformation, align the zero hour background and line images to the 24 hour background image so that the area migration lines are positioned correctly in the 24 hour image. When the line layer has been successfully overlaid, right click to delete the zero hour background layer. The remaining line layer can be used to determine the number of cells that migrated into the scratch area over 24 hours.Then save the new migration image as both a Photoshop and a TIFF or JPEG file. To quantify the number of migrating cells, open the migration image and a program that accepts TIFF or JPEG files and manually count the number of cells located in between and touching the two red lines for both images for each well. Then record the number of migrating cells per image.To determine the area of migration, open the 24 hour image that contains the lines of migration in the imaging software and save the image as migration area image. After saving, click the background layer and right click to delete the layer. Use the brush tool to fill in the area between the scratch lines, with the same color that was used for the lines and click image, mode, and gray scale to change the image to black and white.Save the image and open the migration area image and an appropriate image analysis software program. Click edit, and invert. To determine the area of the line of migration, click image, adjust, and threshold.The black migration area will turn red and the percent area will be indicated in the threshold box. Then record the percent area for the line of migration and confirm that this value is paired with the number of migrating cells for the same image. In this analysis, 46 fibroblasts from non-diabetic hearts and 129 fibroblasts from diabetic hearts migrated during the 24 hour experimental time period.Measurement of the migration area as demonstrated, revealed that the non-diabetic scratch area made up 24.78%of the total area measured, while the diabetic migration scratch area made up 16.77%of the total area measured. Normalizing to the area of migration provides a better and more rigorous assessment of cell migration and negates potential human error. For example, if only the number of migrated fibroblasts are compared, it would appear in this analysis that the number of cells that migrated in this well was twice that of the migrated cells in the second well.When the area of the scratch was used to normalize the data, however, the ratio of the fibroblasts to the migration area was observed to be nearly the same in both wells. Be sure to recapture the area of migration in the 24 hour image, as you captured in the zero hour image. Following this procedure, immunohistochemistry can be conducted to examine protein expression within the cells.We believe this newer approach to the scratch migration assay will allow for more avenues of scientific research to open up that previously were not available.

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5.1K Views.  University of Mississippi. This protocol provides a new method for an old technique, and it will provide a new approach for an large number of basic science researchers. The main advantages of this technique are it's cost effective nature as well as its adaptability to be used by a wider scientific audience. To prepare a migration plate, use a light colored permanent marker to draw a line down the center of the back of each well of a 48 well plate and to draw three hash marks to divide each well into three separate are...