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. Cell physiology. 301, C695-C704 (2011).</li><li>Boyden, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemotactic+effect+of+mixtures+of+antibody+and+antigen+on+polymorphonuclear+leucocytes.">The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes.</a> The Journal of Experimental Medicine. 115, 453-466 (1962).</li><li>Relou, I. A., Damen, C. A., van der Schaft, D. W., Groenewegen, G., Griffioen, A. W. <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+culture+conditions+on+endothelial+cell+growth+and+responsiveness.">Effect of culture conditions on endothelial cell growth and responsiveness.</a> Tissue &amp; Cell. 30, 525-530 (1998).</li><li>Smeets, E. F., von Asmuth, E. J., vander Linden, C. J., Leeuwenberg, J. F., Buurman, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+substrates+for+human+umbilical+vein+endothelial+cell+culture.">A comparison of substrates for human umbilical vein endothelial cell culture.</a> Biotechnic &amp; Histochemistry : Official Publication of the Biological Stain Commission. 67, 241-250 (1992).</li><li>Perry, S. W., Norman, J. P., Barbieri, J., Brown, E. B., Gelbard, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+membrane+potential+probes+and+the+proton+gradient:+a+practical+usage+guide.">Mitochondrial membrane potential probes and the proton gradient: a practical usage guide.</a> BioTechniques. 50, 98-115 (2011).</li></ol>

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><tbody><tr><td>Boyden Chamber&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Corning FluoroBlok Tissue Culture (TC)-treated Inserts, 24 well - 3 &amp;micro;m</td><td>Corning Incorporated</td><td>#351151</td><td></td></tr><tr><td>Corning FluoroBlok Tissue Culture (TC)-treated Inserts, 24 well - 8 &amp;micro;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>Wound&amp;nbsp; 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>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>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>&amp;alpha;-Linolenic acid</td><td>Fluka (Sigma), Steinheim, Germany</td><td>&amp;nbsp;# 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 ...

assessment-endothelial-cell-migration-after-exposure-to-toxic

Universidad de Cartagena UDEC

Research

JoVE Journal

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

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an &#34;in-incubator&#34; video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

Cancer Research

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Bioengineering

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli (UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder.

We developed an in vitro model that mimics an important component of the acute inflammatory response during infection of the bladder with uropathogenic Escherichia coli. The transuroepithelial neutrophil migration assay enables quantitative assessment of human neutrophil migration across bladder epithelia, cultured on permeable supports, in response to bacterial infection or chemoattractant substances.

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. ...

Immunology and Infection

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17- and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 &#181;m pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Neuroscience

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 ...

Biology

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

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.

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 ...

An Introduction to Cell Motility and Migration

Cell motility and migration play important roles in both normal biology and in disease. On one hand, migration allows cells to generate complex tissues and organs during development, but on the other hand, the same mechanisms are used by tumor cells to move and spread in a process known as cancer metastasis. One of the primary cellular machineries that make cell movement possible is an intracellular network of myosin and actin molecules, together known as &#8220;actomyosin&#8221;, which creates a contractile force to pull a cell in different directions.In this video, JoVE presents a historical overview of the field of cell migration, noting how early work on muscle contraction led to the discovery of the actomyosin apparatus. We then explore some of the questions researchers are still asking about cell motility, and review techniques used to study different aspects of this phenomenon. Finally, we look at how researchers are currently studying cell migration, for example, to better understand metastasis.

Cell Motility and Migration, Cell Invasion and Chemo-attraction

Cell motility and migration play important roles in both normal biology and in disease. On one hand, migration allows cells to generate complex tissues ...

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Summary

Explore More Videos

Chapters in this video

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

In vitro Cell Migration and Invasion Assays

Quantitation of Endothelial Cell Adhesiveness In Vitro

A Cost Effective and Adaptable Scratch Migration Assay

An Introduction to Cell Motility and Migration