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>
	<li>Gilbert, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Developmental biology. , (1997).</li><li>Luster, A. D., Alon, R., Von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cell+migration+in+inflammation:+present+and+future+therapeutic+targets.">Immune cell migration in inflammation: present and future therapeutic targets.</a> Nature Immunology. 6, (2005).</li><li>Yahata, Y., 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=A+Novel+Function+of+Angiotensin+II+in+Skin+Wound+Healing+Induction+of+Fibroblast+and+Keratinocyte+Migration+by+Angiotensin+II+via+Heparin-Binding+Epidermal+Growth+Factor+(EGF)-like+Growth+Factor-Mediated+EGF+Receptor+Transactivation.">A Novel Function of Angiotensin II in Skin Wound Healing Induction of Fibroblast and Keratinocyte Migration by Angiotensin II via Heparin-Binding Epidermal Growth Factor (EGF)-like Growth Factor-Mediated EGF Receptor Transactivation.</a> The Journal of Biological Chemistry. 281 (19), 13209-13216 (2006).</li><li>Trepat, X., Chen, Z., Jacobson, K. <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+Single-Cell+Migration.">Cell Migration Single-Cell Migration.</a> Comprehensive Physiology. 2 (4), (2012).</li><li>Brand, S., 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=IL-22+is+increased+in+active+Crohn's+disease+and+promotes+proinflammatory+gene+expression+and+intestinal+epithelial+cell+migration.">IL-22 is increased in active Crohn's disease and promotes proinflammatory gene expression and intestinal epithelial cell migration.</a> American Journal of Physiology: Gastrointestinal and Liver Physiology. 290, 827-838 (2006).</li><li>Chi, Z., Melendez, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Cell+Adhesion+Molecules+and+Immune-Cell+Migration+in+the+Initiation,+Onset+and+Development+of+Atherosclerosis.">Role of Cell Adhesion Molecules and Immune-Cell Migration in the Initiation, Onset and Development of Atherosclerosis.</a> Cell Adhesion &amp; Migration. 1 (4), 171-175 (2007).</li><li>Chen, H., Nalbantoglu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ring+cell+migration+assay+identifies+distinct+effects+of+extracellular+matrix+proteins+on+cancer+cell+migration.">Ring cell migration assay identifies distinct effects of extracellular matrix proteins on cancer cell migration.</a> BMC Research Notes. 7 (183), 1-9 (2014).</li><li>Stewart, J. A., Massey, E. P., Fix, C., Zhu, J., Goldsmith, E. 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A., Laverdet, B., Bont&#233;, F., Desmouli&#232;re, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+and+myofibroblasts+in+wound+healing.">Fibroblasts and myofibroblasts in wound healing.</a> Clinical, Cosmetic and Investigational Dermatology. , 7 (2014).</li><li>Kramer, N., 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+and+invasion+assays.">In vitro cell migration and invasion assays.</a> Mutation Research - Reviews in Mutation Research. 752 (1), 10-24 (2013).</li><li>Even-Ram, S., Yamada, K. M. <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+in+3D+matrix.">Cell migration in 3D matrix.</a> Current Opinion in Cell Biology. (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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Research

JoVE Journal

Biology

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their ...

A Customizable Chamber for Measuring Cell Migration

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between cells, the extracellular matrix, and soluble and non-soluble chemical factors (e.g., chemoattractants). Standard methods for measuring the migration of cells, such as the Boyden chamber assay, work by counting cells on either side of a divider. These techniques are easy to use; however, they offer little geometric modification for different applications. In contrast, microfluidic devices can be used to observe cell migration with customizable concentration gradients of soluble factors1,2. However, methods for making microfluidics based assays can be difficult to learn.
Here, we describe an easy method for creating cell culture chambers to measure cell migration in response to chemical concentration gradients. Our cell migration chamber method can create different linear concentration gradients in order to study cell migration for a variety of applications. This method is relatively easy to use and is typically performed by undergraduate students.
The microchannel chamber was created by placing an acrylic insert in the shape of the final microchannel chamber well into a Petri dish. After this, poly(dimethylsiloxane) (PDMS) was poured on top of the insert. The PDMS was allowed to harden and then the insert was removed. This allowed for the creation of wells in any desired shape or size. Cells may be subsequently added to the microchannel chamber, and soluble agents can be added to one of the wells by soaking an agarose block in the desired agent. The agarose block is added to one of the wells, and time-lapse images can be taken of the microchannel chamber in order to quantify cell migration. Variations to this method can be made for a given application, making this method highly customizable.

This protocol details a customizable method to measure cell migration in response to chemoattractants that may also be used to determine the diffusion rate of a drug out of a polymer matrix.

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, implantation of transgenic cells and tiny optical sensors. However, intravenous microinjections into small animals, which are mostly used in biological and veterinary laboratories, are very difficult and require trained personnel. Herein, we demonstrate a robust and efficient method for the introduction of microparticles into the circulatory system of adult zebrafish (Danio rerio) by injection into the fish kidney. To visualize the introduced microparticles in the vasculature, we propose a simple intravital imaging technique in fish gills. In vivo monitoring of the zebrafish blood pH was accomplished using an injected microencapsulated fluorescent probe, SNARF-1, to demonstrate one of the possible applications of the described technique. This article provides a detailed description of the encapsulation of pH-sensitive dye and demonstrates the principles of the quick injection and visualization of the obtained microcapsules for in vivo recording of the fluorescent signal. The proposed method of injection is characterized by a low mortality rate (0-20%) and high efficiency (70-90% success), and it is easy to institute using commonly available equipment. All described procedures can be performed on other small fish species, such as guppies and medaka.

This article demonstrates the principles of a quick, minimally invasive injection of fluorescent microparticles into the circulatory system of small fishes and the in vivo visualization of the microparticles in fish blood.

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, ...

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 ...

At-Risk Butterfly Captive Propagation Programs to Enhance Life History Knowledge and Effective Ex Situ Conservation Techniques

Improving knowledge of ex situ best practices for at-risk butterflies is important for generating successful conservation and recovery program outcomes. Research on such captive populations can also yield valuable data to address key information gaps about the behavior, life history, and ecology of the target taxa. We describe a protocol for captive propagation of the federally endangered Cyclargus thomasi bethunebakeri that can be used as a model for other at-risk butterfly ex situ programs, especially those in the family Lycaenidae. We further provide a simple and straightforward protocol for recording various life history metrics that can be useful for informing ex situ methodologies as well as adapted for laboratory studies of other lepidoptera.

Here, we present protocols for 1) the laboratory captive propagation of the federally endangered Miami blue butterfly (Cyclargus thomasi bethunebakeri), and 2) assessing basic life history information such as immature development time and number of larval stadia. Both methods can be adapted for use with other ex situ conservation programs.

Improving knowledge of ex situ best practices for at-risk butterflies is important for generating successful conservation and recovery program outcomes. ...

Live Imaging of Microtubule Dynamics in Glioblastoma Cells Invading the Zebrafish Brain

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment failure is mainly due to the invasiveness of GBM cells, which speaks for the need for a better understanding of GBM motile properties. To investigate the molecular mechanism supporting GBM invasion, new physiological models enabling in-depth characterization of protein dynamics during invasion are required. These observations would pave the way to the discovery of novel targets to block tumor infiltration and improve patient outcomes. This paper reports how an orthotopic xenograft of GBM cells in the zebrafish brain permits subcellular intravital live imaging. Focusing on microtubules (MTs), we describe a procedure for MT labeling in GBM cells, microinjecting GBM cells in the transparent brain of 3 days post fertilization (dpf) zebrafish larvae, intravital imaging of MTs in the disseminating xenografts, altering MT dynamics to assess their role during GBM invasion, and analyzing the acquired data.

We report a technique permitting live imaging of microtubule dynamics in glioblastoma (GBM) cells invading a vertebrate brain tissue. Coupling the orthotopic injection of fluorescently tagged GBM cells into a transparent zebrafish brain with high-resolution intravital imaging allows the measurement of cytoskeleton dynamics during in situ cancer invasion.

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment ...

Scalable, Flexible, and Cost-Effective Seedling Grafting

Early-stage seedling grafting has become a popular tool in molecular genetics to study root-shoot relationships within plants. Grafting early-stage seedlings of the small model plant, Arabidopsis thaliana, is technically challenging and time consuming due to the size and fragility of its seedlings. A growing collection of published methods describe this technique with varying success rates, difficulty, and associated costs. This paper describes a simple procedure to make an in-house reusable grafting device using silicone elastomer mix, and how to use this device for seedling grafting. At the time of this publication, each reusable grafting device costs only $0.47 in consumable materials to produce. Using this method, beginners can have their first successfully grafted seedlings in less than 3 weeks from start to finish. This highly accessible procedure will allow plant molecular genetics labs to establish seedling grafting as a normal part of their experimental process. Due to the full control users have in the creation and design of these grafting devices, this technique could be easily adjusted for use in larger plants, such as tomato or tobacco, if desired.

This protocol describes a robust seedling grafting method that requires no prior experience or training and can be executed at a very low cost using materials easily accessible in most molecular biology labs.

Early-stage seedling grafting has become a popular tool in molecular genetics to study root-shoot relationships within plants. Grafting early-stage ...