This work was funded by the German Ministry of Defense (Grant No. M-SAB1-6-A009).

The following protocol describes techniques for the investigation of early endothelial cell migration. The proper cultivation of vascular endothelial cells requires pre-coating of cell culture flasks with gelatin to ensure proper proliferation and maintenance of an endothelial phenotype.
1. Pre-coating of Cell Culture Flasks
<ol>
	<li>Dissolve gelatin in 0.1 M PBS to a final concentration of 0.1%.</li>
	<li>Autoclave the solution with parameters for liquid autoclaving (for details see instructions of the specific autoclave).</li>
	<li>Add sufficient volume of the autoclaved gelatin solution to a sterile cell culture flask (e.g., at least 5 ml for a T25 cell culture flask).</li>
	<li>Transfer the flasks into an incubator (37&#176; C, 5% CO2 is not required but does not interfere) for at least 30 min.</li>
	<li>Remove the remaining gelatin solution. Use the pre-coated flasks subsequently (see cell cultivation) or store under sterile conditions until use.</li>
</ol>
2. Cell Cultivation of Early Endothelial Cells
Note: Embryonic stem cell derived early endothelial cells (EEC) were obtained from differentiated murine embryoid bodies by magnetic-activated cell sorting of the PECAM-1 positive cell fraction as described earlier10,11.
<ol>
	<li>Culture EEC on gelatin-coated cell culture dishes in DMEM supplemented with 15% FCS, 50 U/ml penicillin, 50 U/ml streptomycin, 200 &#181;M L-glutamine, 100 &#181;M &#223;-mercaptoethanol and 1% non-essential amino acids. Handle cells under sterile conditions. Cultivate the cells with 5% CO2 at 37 &#176;C and 95% humidity until sub-confluence (max. 80%).</li>
	<li>At sub-confluence, split the cells at a 1:5 ratio. Note: Detachment of endothelial cells is a critical step.
	<ol>
		<li>Harvest the cells with RT accutase. Remove the media, rinse with PBS and add 1 ml of accutase per 25 cm2.</li>
		<li>Incubate the flask at RT for 2-10 min until the cells have detached. Disperse the cells and transfer them to the desired application. Note: A chemical neutralization of the accutase is not required as it takes place when the seeded cells are stored in the incubator at 37&#160;&#176;C. However, accutase activity can also be decreased by adding DMEM containing FCS.</li>
	</ol>
	</li>
</ol>
3. Boyden Chamber
Note: Boyden chamber assays are performed by using light-opaque polyethylene terephthalate insert systems with 8 &#181;m pore size.
<ol>
	<li>Pre-coat the filter inserts (that fit inside cell culture wells thus creating a Boyden chamber) by adding 500 &#181;l of 0.1% gelatin dissolved in 0.1 M PBS for at least 30 min.</li>
	<li>If cells are to be exposed to toxic chemicals, expose according to the specific experimental design before cell harvesting. Note: Cells were exposed to 12.5 &#181;g chlorambucil/ml DMEM for 24 hr. With regard to the specific experimental design, instructions may vary.</li>
	<li>Harvest EEC and determine the cell number by using a counting cell chamber. Note: Automatic counting devices can be used, but should be used with caution: manual cell counting is more accurate and is highly recommended.</li>
	<li>Add 500 &#181;l cell culture medium into the lower chamber compartment of the Boyden Chamber.</li>
	<li>Add exactly 104 EEC in 500 &#181;l cell culture medium per filter insert in the upper chamber compartment. Eliminate bubbles.</li>
	<li>Incubate the filter inserts in the incubator for exactly 8 hr.</li>
	<li>Rinse with PBS once and replace the medium with 0.5 ml 4% paraformaldehyde in both compartments for 25 min for cell fixation. Wash the filter extensively but at least 3 times with 0.1 M PBS.</li>
	<li>Excise the membranes with a scalpel.</li>
	<li>Mount the membrane between two glass cover slips with mounting medium containing DAPI for nuclear staining. Pay attention to the orientation. Ensure that only cells that have migrated towards the lower compartment side of the membrane are counted.</li>
	<li>Count cells that have migrated towards the lower compartment side of the membrane with a fluorescence microscope at 400X magnification. Do not confuse membrane pores with migrated cells (Figure 1A, 1B). Investigate a reasonable number of biological replicates (at least 3 biological replicates per condition).</li>
</ol>
4. Wound Healing Assay
<ol>
	<li>Depending on the available equipment, carefully choose the cell culture dishes or plates: When using DIC microscopy, avoid plastic surface based culture dishes or well plates but use glass based devices instead. 
	Note: If using phase-contrast microscopy, plastic based dishes can also be used.</li>
	<li>Cultivate EEC in a suitable cell culture device (e.g., 4 cm glass bottom petri dish suitable for live cell imaging) until 80% confluence. Important: do not cultivate the cells to complete confluence.</li>
	<li>Scratch the monolayers with sterile 10 &#181;l pipette tips. Push the tip gently without too much pressure onto the dish surface and move it in a straight line smoothly from one side to the other.Wash the cells twice with 0.1 M PBS to remove detached cells.</li>
	<li>Add a sufficient volume of medium to the culture dish. If applicable, add compounds that should be investigated. 
	Note: 1.5 ml medium containing 15 ng/ml alpha linolenic acid was added.</li>
	<li>Mount the culture dish under a microscope capable of live cell imaging. Ensure 5% CO2, 37 &#176;C and a humidified atmosphere. 
	Note: Humidification is especially important to avoid medium evaporation.</li>
	<li>Acquire time-lapse images over 24 hr at 10 min intervals. Plan for large file sizes. Note: A resolution of 512 x 512 pixels is usually sufficient; however, we recommend using images of at least 1,024 x 1,024.</li>
	<li>Measure the gap width at t = 0 hr and at t = 24 hr using the length tool of the software provided with the microscope or use open-source software (e.g., ImageJ). Note: In general specific image acquisition and analysis software is provided by the manufacturer. Therefore, for technical details regarding the use of the software, refer to the manual.</li>
</ol>
5. Cell Tracking
<ol>
	<li>Depending on the available equipment, carefully choose the cell culture dishes or plates: When using DIC microscopy, avoid plastic surface based culture dishes or well plates but use glass based devices instead. 
	Note: If using phase-contrast microscopy, plastic based dishes can also be used.</li>
	<li>Seed 5 x 104 EEC in supplemented DMEM in a suitable cell culture device (e.g., 24-multiwell plate) and cultivate the cells with 5% CO2 at 37 &#176;C and 95% humidity for 1-2 days.</li>
	<li>When cells have grown up to a 80% confluence, remove the media and culture the cells with new media in the presence of the respective test substances (e.g., 12.5 &#181;g chlorambucil / ml DMEM). Always include control cells (treated with the solvent, e.g., ethanol) at 37 &#176;C for a certain period of time (24 hr, depending on the individual assay system).</li>
	<li>Mount the culture dish under a microscope capable of live cell imaging (37 &#176;C, 5% CO2 and 95% humidity). Acquire time-lapse images over 24 hr at predefined intervals. Acquire images at 10 min intervals.</li>
	<li>Perform manually tracking of EEC by the use of the ImageJ plugin MTrackJ. Choose 10 cells randomly from the field of view and track their movements by adding a data point per point in time using the &#8220;Add&#8221; command of MTrackJ.</li>
</ol>
Note: MTrackJ is available for free at [Meijering, \Mtrackj.&#34; http://www.imagescience.org/meijering/software/mtrackj/ &#8220;] and ImageJ is available at [Rasband, \Imagej.&#34; http://rsbweb.nih.gov/ij/ &#8220;]. A detailed manual about the MTrackJ plugin is available at &#8220;http://www.imagescience.org&#8221;.
6. Live Cell Imaging/Assessment of Mitochondrial Membrane Potential
<ol>
	<li>Cultivate EEC in a suitable cell culture device (e.g., 4 cm glass bottom petri dish suitable for live cell imaging) up to a 80% confluence. 
	Important: Do not cultivate the cells to complete confluence.</li>
	<li>If applicable, expose the cells to chemicals. Note: Cells were exposed to 12.5 &#181;g/ml chlorambucil for 24 hr. With regard to the specific experimental design, instructions may vary.</li>
	<li>Prepare a 10 mM stock solution of tetramethylrhodamine (TMRM) in DMSO. Protect from light. Note: The stock solution can be stored at -20 &#176;C.</li>
	<li>Dilute the stock solution in cell culture medium to a working solution with a concentration of 10 &#181;M (1:1,000 dilution). Protect from light and use as soon as possible. Note: The working solution can be kept at RT for some time (&#62;1 hr), however, preparation of a fresh working solution is highly recommended.</li>
	<li>Add 2 &#181;l of the working solution to 1 ml fresh cell culture medium (loading solution).</li>
	<li>Load the cells by replacing the cell culture medium with the loading solution. Incubate for 15 min at 37 &#176;C, 5% CO2 and humidified atmosphere (incubator). Caution: Almost all fluorescence indicators are exported by living cells over time; therefore avoid prolonged loading or delayed analysis.</li>
	<li>Without washing, place the dish under a microscope suitable for live cell imaging. Important: fluorescence indicators are highly sensitive to light, therefore, avoid unnecessary light exposure.</li>
	<li>Acquire images without changing the acquisition parameters to ensure comparability between different images.</li>
</ol>

<ol>
	<li>Bauer, S. M., Bauer, R. J., Velazquez, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis,+vasculogenesis,+and+induction+of+healing+in+chronic+wounds.">Angiogenesis, vasculogenesis, and induction of healing in chronic wounds.</a> Vascular and Endovascular Surgery. 39, 293-306 (2005).</li><li>Reinke, J. M., Sorg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+repair+and+regeneration.">Wound repair and regeneration.</a> European Surgical Research. Europaische Chirurgische Forschung. Recherches Chirurgicales Europeennes. 49, 35-43 (2012).</li><li>Hart, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation.+1:+Its+role+in+the+healing+of+acute+wounds.">Inflammation. 1: Its role in the healing of acute wounds.</a> Journal of Wound Care. 11, 205-209 (2002).</li><li>Liu, Z. J., Hyperoxia Velazquez, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=endothelial+progenitor+cell+mobilization,+and+diabetic+wound+healing.">endothelial progenitor cell mobilization, and diabetic wound healing.</a> Antioxidants &amp; Redox Signaling. 10, 1869-1882 (2008).</li><li>Gallagher, K. A., Goldstein, L. J., Thom, S. R., Velazquez, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperbaric+oxygen+and+bone+marrow-derived+endothelial+progenitor+cells+in+diabetic+wound+healing.">Hyperbaric oxygen and bone marrow-derived endothelial progenitor cells in diabetic wound healing.</a> Vascular. 14, 328-337 (2006).</li><li>Kehe, K., Balszuweit, F., Steinritz, D., Thiermann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+toxicology+of+sulfur+mustard-induced+cutaneous+inflammation+and+blistering.">Molecular toxicology of sulfur mustard-induced cutaneous inflammation and blistering.</a> Toxicology. 263, 12-19 (2009).</li><li>Schmidt, 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=Nitrogen+mustard+(Chlorambucil)+has+a+negative+influence+on+early+vascular+development.">Nitrogen mustard (Chlorambucil) has a negative influence on early vascular development.</a> Toxicology. 263, 32-40 (2009).</li><li>Steinritz, D., 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=Effect+of+N-acetyl+cysteine+and+alpha-linolenic+acid+on+sulfur+mustard+caused+impairment+of+in+vitro+endothelial+tube+formation.">Effect of N-acetyl cysteine and alpha-linolenic acid on sulfur mustard caused impairment of in vitro endothelial tube formation.</a> Toxicological Sciences. 118, 521-529 (2010).</li><li>Steinritz, D., 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=Chlorambucil+(nitrogen+mustard)+induced+impairment+of+early+vascular+endothelial+cell+migration+-+Effects+of+alpha-linolenic+acid+and+N-acetylcysteine.">Chlorambucil (nitrogen mustard) induced impairment of early vascular endothelial cell migration - Effects of alpha-linolenic acid and N-acetylcysteine.</a> Chemico-biological Interactions. 219C, 143-150 (2014).</li><li>Schmidt, 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=Influence+of+endostatin+on+embryonic+vasculo-+and+angiogenesis.">Influence of endostatin on embryonic vasculo- and angiogenesis.</a> Developmental Dynamics : an Official Publication of the American Association of Anatomists. 230, 468-480 (2004).</li><li>Dainiak, M. B., Kumar, A., Galaev, I. Y., Mattiasson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+cell+separations.">Methods in cell separations.</a> Advances in Biochemical Engineering/Biotechnology. 106, 1-18 (2007).</li><li>Wang, 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=Regulation+of+VEGF-induced+endothelial+cell+migration+by+mitochondrial+reactive+oxygen+species.">Regulation of VEGF-induced endothelial cell migration by mitochondrial reactive oxygen species.</a> American Journal of Physiology. 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The authors have nothing to disclose.

Dermal exposure to toxic chemicals often results in severe wound healing disorder. The underlying mechanisms are largely unknown. Wound healing is a complex process that consists of different phases (hemostasis, inflammation, proliferation and remodeling). Cell migration is involved in every phase, however, it is of utmost importance for the formation of the granulation tissue. Here, new blood vessels are formed either by angio- or vasculogenesis.
Both processes require unaffected migration of...

Cell migration is important in many physiological and pathophysiological processes including development, various diseases, and wound healing after skin injury.
Following skin injury, inflammation removes damaged or necrotic tissue and granulation drives preliminary wound closure and allows formation of a network of new capillaries through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vesicles)1-3. Both vasculo- and angiogenesis require migration of endothelial cells. The growing network of blood vessels is essential to transport oxygen and nutrients to proliferating keratinocytes which ultimately undergo keratinization, form a new epithelium and provide wound closure.
Impaired migration of endothelial cells is an underlying cause of wound healing disorder4,5. Thus, methods to assess migration of early endothelial cells are required to explore the pathophysiology of cell migration disorders and to identify novel strategies for therapeutic intervention.
Dermal exposure to alkylating agents (e.g., sulfur and nitrogen mustards) causes wound healing disorder6. Such compounds were used as chemical warfare agents in several conflicts in the 20th century and remain reason for strong concern due to existing stockpiles in politically unstable regions and the relatively simple synthesis. Although sulfur mustard was first synthesized in 1822, the molecular and clinical pathology of SM exposure is not understood in detail and no antidote for SM exposure has been identified.
Several studies have been conducted to understand and to model impaired wound healing after SM exposure and to test for potential candidate compounds capable of reserving that effect. Schmidt et al. (2009) tested the effect of chlorambucil, an alkylating compound with properties similar to SM in mouse embryoid body models and found a dramatic, sometimes more than 99% reduction in vessel formation7. This adverse effect was most pronounced at a stage of development which, under physiological conditions, is dominated by the proliferation and migration of vascular endothelial precursor cells. Thus, these cells were identified to be particularly sensitive to alkylating agents. Steinritz et al. (2010) tested scavengers of reactive oxygen species (ROS), in particular, N-acetylcysteine (NAC) and alpha linolenic acid (ALA) for their ability to reduce SM toxicity in mouse embryoid body models and in particular, to restore vessel formation8. Temporary protective effects were observed, indicating that excessive ROS formation was likely to contribute to the adverse effects of SM on wound healing. These effects were not permanent and the two candidate compounds may not be capable of restoring vessel formation and wound healing in the long term8. However, those experiments were conducted in a complex 3D model which did allow investigation of cell migration. Thus, we subsequently tested NAC and ALA for beneficial effects on cell migration of EEC that have a key role in the process of vessel formation9.
Moreover, there is evidence that cell polarity is required for cell migration. Mitochondrial dysfunction leading to ROS formation was shown to impair cell polarity and may thus adversely affect cell migration. Therefore, live cell imaging with regard to mitochondrial function was performed and the effects of ROS scavengers were examined. The following protocol describes general requirements for the cultivation of EEC, the Boyden chamber assay, the wound healing assay including cell tracking analysis and the use of TMRM for assessment of mitochondrial function in detail. Important aspects of experimental protocols for EEC cultivation and migration are highlighted.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Boyden Chamber&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corning FluoroBlok Tissue Culture (TC)-treated Inserts, 24 well - 3 &#181;m</td>
			<td>Corning Incorporated</td>
			<td>#351151</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning FluoroBlok Tissue Culture (TC)-treated Inserts, 24 well - 8 &#181;m</td>
			<td>Life Sciences</td>
			<td>#351152</td>
			<td></td>
		</tr>
		<tr>
			<td>(for use with Falcon Insert 24 well Companion Plate (353504)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wound&#160; healing assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom dishes</td>
			<td>Word Precision Instruments, Inc.</td>
			<td>#FD35-100</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Assessment of mitochondrial potential</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TMRM (tetramethylrhodamine methyl ester)</td>
			<td>Life Technologies</td>
			<td>#T669</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>PAA, Pasching, Austria</td>
			<td># L11-007</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-Linolenic acid</td>
			<td>Fluka (Sigma), Steinheim, Germany</td>
			<td>&#160;# L2376</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorambucil</td>
			<td>Fluka (Sigma), Steinheim, Germany</td>
			<td># 23125</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich, Steinheim, Germany</td>
			<td># G2500-100G</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of endothelial cell migration after exposure to toxic chemicals

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels &#8211; either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention.
We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.

Dermal exposure to alkylating agents provokes erythema, blister formation and dermal ulceration that is associated with a wound healing disorder. Wound healing requires angio- and vasculogenesis which are based on migration of endothelial cells. Quantitative migration can be assessed by use of the Boyden chamber assay. As shown in Figure 1C exposure of EEC to the alkylating agent chlorambucil resulted in a significant decrease in cell migration9. Addition of the ROS-scavenger alpha linolenic a...

Watch this Scientific Journal Video about Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals at JoVE.com

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

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Developmental Biology

8.6K Views.  Bundeswehr Institute of Pharmacology and Toxicology. Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

Video: Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.

Zebrafish keratocytes migrate in cell sheets from explants and provide an in vitro model for the study of the mechanisms of collective cell migration in the context of epithelial wound healing. These protocols detail an effective way to establish primary explant cultures for use in collective cell migration assays.

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

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

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.

The capacity to migrate is a key function of many different cell types, including mesenchymal stromal cells (MSCs). However, quantifying alterations in migratory capacity after damage is challenging. This protocol describes an easily adaptable migration assay that uses rigorous statistics to quantify changes in MSC migratory capacity after damage.

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including embryogenesis and development, wound healing, tumor growth and metastasis, and ocular and cardiovascular diseases. Effective in vitro models that recapitulate the biology of angiogenesis are needed to appropriately study this process and identify mechanisms of regulation that can be ultimately targeted for novel therapeutic strategies. The bead angiogenesis assay has been previously demonstrated to recapitulate the multiple stages of endothelial sprouting in vitro. However, a limitation of this assay is a lack of endothelial &#8211; mural cell interactions, which are key to the molecular and phenotypic regulation of endothelial cell function in vivo. The protocol given here presents a methodology for the incorporation of mural cells into the bead angiogenesis assay and demonstrates a tight association of endothelial cells and pericytes during sprouting in vitro. The protocol also details a methodology for effective silencing of target genes using siRNA in endothelial cells for mechanistic studies. Altogether, this protocol provides an in vitro assay that more appropriately models the diverse cell types involved in sprouting angiogenesis, and provides a more physiologically-relevant platform for therapeutic assessment and novel discovery of mechanisms of angiogenesis regulation.

This protocol presents a novel in vitro bead assay that more appropriately models the process of in vivo sprouting angiogenesis by incorporating pericytes. This modification enables the bead assay to more faithfully recapitulate the heterotypic cellular interactions between endothelial cells and mural cells that are critical for angiogenesis.

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs)

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected tissues via a well-characterized cascade of pro-adhesive receptors which are upregulated on the leukocyte cell surface upon the inflammatory trigger. Inflammatory responses differ between individuals in the population and the genetic background can contribute to these differences. Human induced pluripotent stem cells (hiPSCs) have been shown to be a reliable source of ECs (hiPSC-ECs), thus representing an unlimited source of cells that capture the genetic identity and any genetic variants or mutations of the donor. hiPSC-ECs can therefore be used for modeling inflammatory responses in donor-specific cells. Inflammatory responses can be modeled by determining leukocyte adhesion to the hiPSC-ECs under physiological flow. This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells hiPSC-ECs

This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected ...

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration ...