Name: a cost effective and adaptable scratch migration assay
Uploaded: 2020-06-30T15:00:00
Description: Watch this Scientific Journal Video about A Cost Effective and Adaptable Scratch Migration Assay at JoVE.com

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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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 ...

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