Name: evaluation of the cell invasion and migration process: a comparison of the video microscope-based scratch wound assay and the boyden chamber assay
Uploaded: 2017-11-17T13:00:00
Description: Watch this Scientific Journal Video about Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay at JoVE.com

These techniques were developed with the support of LabEx PRIMES (ANR-11-LABX-0063), the Contrat Plan-Etat-Region (CPER) within the scientific framework of ETOILE (CPER 2009-2013), and Lyric Grant INCa-DGOS-4664.

NOTE: The Boyden chamber and scratch assays without inclusion of the ECM are referred to as the migration assay, and the same assays with the ECM is referred to as the invasion assay.
1. Boyden Chamber Assay
NOTE: This protocol is adapted for the SQ20B cell line, which is derived from a recurrent Head and Neck Squamous Cell Carcinoma (HNSCC) laryngeal cancer and was obtained from John Little (Boston, MA, USA).
Day 1
<ol...

<ol>
	<li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-cell+invasion+and+migration:+diversity+and+escape+mechanisms.">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nat Rev Cancer. 3 (5), 362-374 (2003).</li><li>Moncharmont, C., 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=Radiation-enhanced+cell+migration/invasion+process:+A+review.">Radiation-enhanced cell migration/invasion process: A review.</a> Crit Rev Oncol Hematol. , (2014).</li><li>Burdsal, C. A., Damsky, C. H., Pedersen, R. 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+role+of+E-cadherin+and+integrins+in+mesoderm+differentiation+and+migration+at+the+mammalian+primitive+streak.">The role of E-cadherin and integrins in mesoderm differentiation and migration at the mammalian primitive streak.</a> Development. 118 (3), 829-844 (1993).</li><li>Chen, C., Zimmermann, M., Tinhofer, I., Kaufmann, A. M., Albers, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-to-mesenchymal+transition+and+cancer+stem(-like)+cells+in+head+and+neck+squamous+cell+carcinoma.">Epithelial-to-mesenchymal transition and cancer stem(-like) cells in head and neck squamous cell carcinoma.</a> Cancer Lett. 338 (1), 47-56 (2013).</li><li>Nelson, A. R., Fingleton, B., Rothenberg, M. L., Matrisian, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinases:+biologic+activity+and+clinical+implications.">Matrix metalloproteinases: biologic activity and clinical implications.</a> J Clin Oncol. 18 (5), 1135-1149 (2000).</li><li>Smith, A., Teknos, T. N., Pan, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+to+mesenchymal+transition+in+head+and+neck+squamous+cell+carcinoma.">Epithelial to mesenchymal transition in head and neck squamous cell carcinoma.</a> Oral Oncol. 49 (4), 287-292 (2013).</li><li>Chen, H. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boyden+chamber+assay.">Boyden chamber assay.</a> Meth Mol Biol. 294, 15-22 (2005).</li><li>Rodriguez, L. G., Wu, X., 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=Wound-healing+assay.">Wound-healing assay.</a> Meth Mol Biol. 294, 23-29 (2005).</li><li>Piaton, E., 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=Technical+recommendations+and+best+practice+guidelines+for+May-Gr&#252;nwald-Giemsa+staining:+literature+review+and+insights+from+the+quality+assurance.">Technical recommendations and best practice guidelines for May-Gr&#252;nwald-Giemsa staining: literature review and insights from the quality assurance.</a> Ann Path. 35 (4), 294-305 (2015).</li><li>Moncharmont, C., 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=Targeting+a+cornerstone+of+radiation+resistance:+cancer+stem+cell.">Targeting a cornerstone of radiation resistance: cancer stem cell.</a> Cancer lett. 322 (2), 139-147 (2012).</li><li>Moncharmont, C., 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=Carbon+ion+irradiation+withstands+cancer+stem+cells’+migration/invasion+process+in+Head+and+Neck+Squamous+Cell+Carcinoma+(HNSCC).">Carbon ion irradiation withstands cancer stem cells’ migration/invasion process in Head and Neck Squamous Cell Carcinoma (HNSCC).</a> Oncotarget. 7 (30), 47738-47749 (2016).</li><li>Gilormini, M., Wozny, A. -. S., Battiston-Montagne, P., Ardail, D., Alphonse, G., Rodriguez-Lafrasse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+a+Head+and+Neck+Squamous+Cell+Carcinoma+Subpopulation+Having+Stem+Cell+Characteristics.">Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics.</a> J Vis Exp: JoVE. (111), (2016).</li></ol>

We report here two different modalities to study the cell invasion and migration process. The analysis of this process is important to understanding the factors involved in metastatic recurrence, which might be explained by increased motility of a subpopulation of cancer cells called cancer stem cells10,11.
The Boyden chamber experiment is one the most frequently used techniques to explore cell invasion and migration. One advantage of ...

Cell invasion and migration are involved in the dissemination of cancer cells, which is the main cause of resistance to treatment1 and can lead to locoregional or metastatic recurrence after cancer treatment2. The epithelial-mesenchymal transition (EMT) is the initial process of cell invasion-migration in which cancer cells switch from an epithelial to a mesenchymal phenotype. E-Cadherin is an extracellular marker of the epithelial phenotype3, and increased expression of N-cadherin and vimentin is characteristic of the mesenchymal phenotype4. Migration also depends on the intrinsic capacity of cancer cells to invade the extracellular matrix (ECM) through the action of matrix metalloproteases5.
This invasion&#8211;migration mechanism has been described for cancer at many locations, particularly in head and neck cancer6. Many researchers have focused on the migration and invasion processes to understand better how cancer cells disseminate in the hope that this knowledge will lead to new treatment strategies. It is crucial that these studies are performed using reliable and reproducible assays.
In vitro analysis of cell motility can be challenging. Developed many years ago, the Boyden chamber assay is considered to be the standard for invasion&#8211;migration analysis7. However, it is time consuming and is often inaccurate. A second test is the wound-healing assay8, which involves making a scratch on a cell monolayer culture and capturing images of cell invasion and migration at fixed time intervals. This technique has been criticized widely because of the large variations between the results of two successive tests. However, the application of modern technologies, especially in microscopy, has improved the reproducibility of the scratch wound assay. Video microscopes can be easily introduced in incubators and can generate real-time images of cell migration. These devices record microscopic data and provide automatic analysis of wound cell confluence over time. The aim of this paper is to describe the Boyden chamber assay and the optimized scratch wound assay, and to discuss the advantages and weaknesses of each approach.

<table width="853">
	<tbody>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">Fetal Calf Serum Gold</td>
			<td style="width:117px;">GE Healthcare</td>
			<td style="width:181px;">A15-151</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">Hydrocortisone water soluble</td>
			<td style="width:117px;">Sigma-Aldrich</td>
			<td style="width:181px;">H0396-100MG</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">Penicillin/Streptomycin 100 X</td>
			<td style="width:117px;">Dominique Dutscher</td>
			<td style="width:181px;">L0022-100</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">DMEM</td>
			<td style="width:117px;">Gibco</td>
			<td style="width:181px;">61965-026</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">F12 Nut Mix (1X) + GlutaMAX-I</td>
			<td style="width:117px;">Gibco</td>
			<td style="width:181px;">31765-027</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">EGF</td>
			<td style="width:117px;">Promega</td>
			<td style="width:181px;">G5021</td>
			<td style="width:339px;">The solution must be prepared just before use because it is very unstable</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">Z1 coulter particle</td>
			<td style="width:117px;">Beckman Coulter</td>
			<td style="width:181px;">6605698</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">Optical microscope</td>
			<td style="width:117px;">Olympus&#160;</td>
			<td style="width:181px;">CKX31</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:216px;height:63px;">SQ20B cell line</td>
			<td style="width:117px;">Gift from the John Little&#8217;s Laboratory</td>
			<td style="width:181px;">-</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">Wound Maker</td>
			<td style="width:117px;">Essen Bioscience</td>
			<td style="width:181px;">4494</td>
			<td style="width:339px;">Store in safe and dry place</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">96-well ImageLock Plate</td>
			<td style="width:117px;">Essen Bioscience</td>
			<td style="width:181px;">4379</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">CoolBox 96F System with CoolSink 96F</td>
			<td style="width:117px;">Essen Bioscience</td>
			<td style="width:181px;">1500-0078-A00</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">CoolBox with M30 System</td>
			<td style="width:117px;">Essen Bioscience</td>
			<td style="width:181px;">1500-0078-A00</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">Boyden Insert</td>
			<td style="width:117px;">Dominique Dutcher</td>
			<td style="width:181px;">353097</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">Boyden Coated Insert</td>
			<td style="width:117px;">Dominique Dutcher</td>
			<td style="width:181px;">354483</td>
			<td style="width:339px;">Store at -20 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">Companion 24-well Plate</td>
			<td style="width:117px;">Dominique Dutcher</td>
			<td style="width:181px;">353504</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">BD Matrigel standard</td>
			<td style="width:117px;">BD BioScience</td>
			<td style="width:181px;">BD 354234</td>
			<td style="width:339px;">Store at -20&#176;C.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">RAL 555 Staining Kit</td>
			<td style="width:117px;">RAL Diagnostics</td>
			<td style="width:181px;">&#160;361550</td>
			<td style="width:339px;">Store in safe and dry place</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">Microcentrifuge tubes</td>
			<td style="width:117px;">Eppendorf</td>
			<td style="width:181px;">33511</td>
			<td style="width:339px;"></td>
		</tr>
	</tbody>
</table>

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.

We report here two different methods to analyze cell invasion and migration. Figure 1 shows the Boyden chamber experiment. The inserts are placed into a companion plate with chemoattractant medium, and the cells are seeded in CM. The membrane can be uncoated (migration assay) or coated (invasion assay). Cells are seeded into the upper chamber in s-CM. The lower chamber is filled with CM as a chemoattractant. The cells are fixed before the doubling time.
<...

Watch this Scientific Journal Video about Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay at JoVE.com

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

Research

JoVE Journal

Cancer Research

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

Using the Dot Assay to Analyze Migration of Cell Sheets

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...