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>
	<li>Cell Seeding
	<ol>
		<li>Seed 2 106 SQ20B cells in 12 mL of culture medium (CM) in a 175 cm2 flask 72 h before day one and allow the cells to grow to 80% cell confluence.
		<ol>
			<li>To prepare CM for SQ20B cells, supplement Dulbecco&#39;s modified Eagle medium (DMEM) with 10% fetal calf serum (FCS), 0.04 mg/mL hydrocortisone, 100 U/mL penicillin, and 0.1 g/L streptomycin.</li>
		</ol>
		</li>
		<li>Under a laminar flow hood, trypsinize the cells. Remove the medium, wash the cells with sterile phosphate-buffered saline (PBS), and add 2.5 mL of trypsin ethylenediaminetetraacetic acid (EDTA) (0.5 g/L). Incubate the cells for 5 min at 37 &#176;C and then stop the reaction by adding 12.5 mL of CM warmed to 37 &#176;C. Count the number of cells using a cell counter.</li>
		<li>Seed the cells in a 25 cm2 flask at a cell density of 6 105 cells for each condition to be evaluated. Incubate the cells for 24 h in 3 mL of CM.</li>
	</ol>
	</li>
</ol>
Day 2
<ol start="2">
	<li>Cell starvation and preparation of coated chambers
	<ol>
		<li>Starve the cells by replacing the medium in each flask with 3 mL of low-fetal bovine serum (FBS) medium using 0.1% bovine serum albumin (BSA) instead of FBS. Starve the cells for 24 h in the incubator.
		<ol>
			<li>To prepare starvation CM (s-CM), supplement DMEM with 0.1% BSA, 0.04 mg/mL hydrocortisone, 100 U/mL penicillin, and 0.1 g/L streptomycin.</li>
		</ol>
		</li>
		<li>For the migration assay, go to step 1.3.</li>
		<li>For the invasion assay, 12 h before day 3, prepare the coated Boyden chambers by adding 500 &#956;L of s-CM to each coated chamber. Use commercially prepared coated Boyden chambers and keep them at 4 &#176;C before use. Place each Boyden chamber in the companion plate and set it in the incubator at 37 &#176;C overnight.</li>
	</ol>
	</li>
</ol>
Day 3
<ol start="3">
	<li>Cell seeding in the Boyden chamber
	<ol>
		<li>Use the companion plate to prepare the chemoattractant.Fill each well of the 24-well companion plate with 750 &#956;L of complete medium with 10% FBS, 0.04 mg/mL hydrocortisone, 100 U/mL penicillin, and 0.1 g/L streptomycin.
		<ol>
			<li>Adapt the chemoattractant for each cell line and each treatment condition. To prepare the chemoattractant CM (ca-CM), supplement DMEM with 10% FCS, 0.4 mg/mL hydrocortisone, 100 U/mL penicillin, and 0.1 g/L streptomycin.</li>
		</ol>
		</li>
		<li>Transfer the upper chamber into the prefilled companion plate, taking care to avoid bubbles.</li>
		<li>For the invasion assay, carefully remove 450 &#956;L of CM from each Boyden chamber.</li>
		<li>Under a laminar flow hood, trypsinize the cells. Remove the medium, wash the cells with sterile PBS, add 0.5 mL of trypsin EDTA (0.5 g/L), and then incubate the cells for 5 min at 37 &#176;C. Stop the reaction by adding CM warmed to 37 &#176;C. Count the number of cells using a cell counter.</li>
		<li>Seed 3 104 SQ20B cells in 500 &#956;L of 0.1% BSA medium, giving a final dilution of 6 104 cells/mL. 
		NOTE: The cell concentration should be adapted for each cell line.</li>
		<li>Place the plate into the incubator at 37 &#176;C for 24 h.</li>
	</ol>
	</li>
</ol>
Day 4
<ol start="4">
	<li>Cell fixation and staining
	<ol>
		<li>Fix the cells before the doubling time specific to that cell line. Fix SQ20B cells before 24 h.</li>
		<li>Remove each insert from the companion plate and carefully remove the cells from the upper chamber using a cotton swab. It is particularly important to remove all cells located in the upper chamber.</li>
		<li>Fix and stain each insert individually to obtain May-Grunwald Giemsa coloration9 using the staining kit provided. Alternatively, use 4% paraformaldehyde in another companion plate for a 30 min fixation.</li>
		<li>Keep the inserts in an empty 24-well companion plate under a laboratory hood to dry each chamber.</li>
	</ol>
	</li>
</ol>
Day 5
<ol start="5">
	<li>Microscopic analysis
	<ol>
		<li>Insert the companion plate with the inserts onto a 20 phase-contrast microscope and count each migrated cell on the lower part of the membrane. Optimize the right focus position by moving the objective from the bottom to the top, and stop the position on the bottom part of the membrane.</li>
		<li>Calculate the ratio between the numbers of migrated cells and seeded cells. Repeat each count for each treatment condition in triplicate.</li>
	</ol>
	</li>
</ol>
2. Scratch Wound Assay: Cell Migration
NOTE: Instructions must be adapted for each type of cell.
Day 1
<ol>
	<li>Cell Seeding
	<ol>
		<li>Seed 2 x 106 SQ20B cells in 12 mL of CM in a 175 cm2 flask 72 h before day one and allow the cells to grow to 80% cell confluence.</li>
		<li>Under a laminar flow hood, trypsinize the cells. Remove the medium, wash the cells with sterile PBS, add 2.5 mL of trypsin EDTA (0.5 g/L), and incubate the cells for 5 min at 37 &#176;C. Stop the reaction by adding 12.5 mL of CM warmed to 37 &#176;C. Count the number of cells using a cell counter.</li>
		<li>Generate a prediluted sample at a cell density of 4 x 105/mL, giving a final density of 4 104 cells per 100 &#956;L in each well. This concentration must be adapted for each cell line and should be in the range of 1 x 104 to 6 104 cells.</li>
		<li>Seed cells into each well of a 96-well plate by depositing 100 &#956;L of the solution prepared in step 2.1.2.</li>
		<li>Leave the plate at room temperature for 5 min to disperse the cells evenly on the bottom of the wells.</li>
		<li>Place the plate into the incubator and allow the cells to adhere to the plate for 12 to 16 h (maximum) at 37 &#176;C.</li>
	</ol>
	</li>
</ol>
Day 2
<ol start="2">
	<li>Scratch wound assay
	<ol>
		<li>Remove the plates prepared in step 2.1 from the incubator.</li>
		<li>Under the hood, make a wound using the commercial wounding device according to the manufacturer&#39;s protocol.</li>
		<li>Immediately remove the medium from each well using a pipette with an adapted conical tip, taking care not to touch the wound.</li>
		<li>Wash the cells twice by repeating the aspiration and using 100 &#956;L of CM warmed to 37 &#176;C for each well.</li>
		<li>Remove the CM using a pipette with an adapted conical tip after the washing steps.</li>
		<li>Add 100 &#956;L of medium specific for each treatment condition to each well.</li>
		<li>Try to avoid creating any bubbles in the wells. A reverse-pipetting technique may be helpful here. Alternatively, remove bubbles with a syringe needle.</li>
		<li>Place the plate into the adapted rack of the video microscope.</li>
		<li>To improve the image quality, allow the plate to warm for at least 15 min before the first scan to avoid condensation on the lower side of the plate.</li>
		<li>Program the schedule of scans, using the video microscope software at one image per well. A maximum 2-h interval is required for an invasion-migration experiment. If the objective of the experiment is to produce a video, an interval of 30 min maximum is preferred.</li>
		<li>At the end of the experiment, wash the wounding device carefully and follow each of the four washing steps indicated by the manufacturer.</li>
	</ol>
	</li>
</ol>
Days 3, 4, and 5
<ol start="3">
	<li>Analysis of migration using the video microscope
	<ol>
		<li>For a minimum of 24 h and up to 5 days, monitor and check the cell migration.</li>
		<li>Use an appropriate cell mask adapted for each cell type to analyze cell migration. To obtain a cell mask adapted for each cell line, generate a cell processing definition from the software using a specific cell image collection.</li>
		<li>Trace the curves and export the data to a spreadsheet, which can be used to analyze and compare the results.</li>
	</ol>
	</li>
</ol>
3. Scratch Wound Assay: Cell Invasion
NOTE: Instructions must be adapted for each type of cell.
Day 1
<ol>
	<li>Cell Seeding
	<ol>
		<li>Use the same protocol for cell seeding as described in step 2.1.</li>
	</ol>
	</li>
</ol>
Day 2
<ol start="2">
	<li>Preparing the ECM
	<ol>
		<li>Defrost the matrix for at least 12 h before use at 4 &#176;C. Make sure that no aggregates are visible. If aggregates are visible, keep the matrix at 4 &#176;C for a longer period until the aggregates disappear. Keep the matrix on ice. Use precooled pipette tips. 
		NOTE: The matrix will solidify if not kept at 4 &#176;C.</li>
		<li>Chill the microcentrifuge tubes on ice for 5 min.</li>
		<li>Take CM cooled to 4 &#176;C from the refrigerator and dilute the matrix in the precooled microcentrifuge tubes to obtain a final concentration of 300 &#956;g/mL.</li>
		<li>Return the microcentrifuge tubes with prediluted matrix to the refrigerator at 4 &#176;C. 
		NOTE: A cooling rack adapted for microcentrifuge tubes is helpful for maintaining the tubes at 4&#176;C throughout the experiment.</li>
	</ol>
	</li>
</ol>
Day 2
<ol start="3">
	<li>Scratch Wound Assay and Treatment of the ECM
	<ol>
		<li>Remove the plate prepared in step 3.1 from the incubator.</li>
		<li>Under the hood, make the wound using the commercial wounding device according to the manufacturer&#39;s protocol.</li>
		<li>Immediately remove the medium from each well using a pipette with an adapted conical tip taking care not to touch the wound.</li>
		<li>Wash the cells twice by repeating the aspiration procedure and using 100 &#956;L of CM warmed to 37 &#176;C.</li>
		<li>After the second wash, place the plate on the 4 &#176;C incubator to equilibrate its temperature for 5 min.</li>
		<li>Remove the cold medium from each well with the aspiration pipette with a conical tip, taking care to pipette carefully from the edge and to avoid touching the wound.</li>
		<li>Add 50 &#956;L of prediluted matrix to each well using precooled conical tips at 4 &#176;C.</li>
		<li>Place the plate in the incubator at 37 &#176;C for 30 min. 
		NOTE: A prewarmed support rack at 37 &#176;C is helpful to accelerate the warming of the 96-well plate.</li>
		<li>Remove the plate from the incubator and add 100 &#956;L of the adapted CM to each well as indicated for each treatment condition.</li>
		<li>Try to avoid creating any bubbles in the wells. A reverse-pipetting technique may be helpful here. Alternatively, remove bubbles with a syringe needle.</li>
		<li>Place the plate into the adapted rack in the video microscope.</li>
		<li>To improve the image quality, allow the plate to warm for at least 15 min before the first scan to avoid condensation on the lower side of the plate.</li>
		<li>Program the schedule of scans, using the video microscope software at one image per well, in the Scratch Wound scan mode. A maximum 2-h interval in required for an invasion-migration experiment. If the objective of the experiment is to produce a video, an interval of 30 min maximum is preferred.</li>
		<li>At the end of the experiment, wash the wounding device carefully and follow each of the four washing steps indicated by the manufacturer.</li>
	</ol>
	</li>
</ol>
Days 3, 4, and 5
<ol start="4">
	<li>Analysis of cell invasion using a video microscope.
	<ol>
		<li>Repeat step 2.3.</li>
	</ol>
	</li>
</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>

The authors have nothing to disclose.

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 this approach is its reproducibility, although this technique is highly dependent on the experimenter&#39;s expertise. Cell counting is manual and can vary between experimenters. Many critical steps must be standardized and followed with care, such as ensuring the use of the appropriate chemoattractant and starvation medium specific to each cell line. Removing cells from the upper membrane with a cotton swab is also critical to ensuring optimal results and avoiding the suboptimal results shown in Figure 2A. If this assay is used to evaluate cell motility, invasion, and migration capacities through a three-dimensional (3D) porous membrane, it is not possible to track cells through a time-lapse experiment. Moreover, commercial inserts with a porous membrane are often expensive.
The video microscope-based scratch wound assay presented here is a robust technique to evaluate cell migration and invasion. The assay is performed with a mechanical wound maker and provides a reproducible comparison between treatment conditions. The video microscopic analysis can be used to generate precise quantitative data for wound healing and cell confluence through a time-lapse experiment. Time-to-time analysis of different treatment conditions can be corroborated by microscopic observations. This analysis integrates cell proliferation during the assay and takes into account the cell-doubling time. Video or raw data content can be exported to obtain a highly visual representation of the results, as shown in the cell migration curves in Figure 3B.
Many critical steps are required to obtain interpretable results. The optimal conditions for cell seeding are dependent on the cell line and must be tested with different dilutions to obtain 90% cell confluence in the wells. To obtain a linear wound, cell plating must not exceed 16 h. The washing step must be done twice and rapidly to avoid introducing cell debris into the wound that could alter the results. For the invasion scratch wound, proper care of the ECM is also a critical step. The cooling rates must be constant, and the ECM should be manipulated with chilled pipette tips and maintained at 4 &#176;C.
These techniques have many advantages, which are summarized in Table 1. Adherent cells are needed for this technique. Scratching causes mechanical injury to the cells, which causes release of cellular contents into the surrounding medium and may influence the migration process. The scratch wound assay provides an estimate of 2D cell motility but not of cell chemotaxis, which cannot be studied using this technique. We have recently developed a video microscopic chemotaxis assay using a commercial plating system. This assay needs cells with a high migration ability, such as cancer stem cells, as described previously12. This new technique may become the gold standard for invasion-migration assays in the future.

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><tbody><tr><td>Fetal Calf Serum Gold</td><td>GE Healthcare</td><td>A15-151</td><td></td></tr><tr><td>Hydrocortisone water soluble</td><td>Sigma-Aldrich</td><td>H0396-100MG</td><td></td></tr><tr><td>Penicillin/Streptomycin 100 X</td><td>Dominique Dutscher</td><td>L0022-100</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>61965-026</td><td></td></tr><tr><td>F12 Nut Mix (1X) + GlutaMAX-I</td><td>Gibco</td><td>31765-027</td><td></td></tr><tr><td>EGF</td><td>Promega</td><td>G5021</td><td>The solution must be prepared just before use because it is very unstable</td></tr><tr><td>Z1 coulter particle</td><td>Beckman Coulter</td><td>6605698</td><td></td></tr><tr><td>Optical microscope</td><td>Olympus&amp;nbsp;</td><td>CKX31</td><td></td></tr><tr><td>SQ20B cell line</td><td>Gift from the John Little&amp;rsquo;s Laboratory</td><td>-</td><td></td></tr><tr><td>Wound Maker</td><td>Essen Bioscience</td><td>4494</td><td>Store in safe and dry place</td></tr><tr><td>96-well ImageLock Plate</td><td>Essen Bioscience</td><td>4379</td><td></td></tr><tr><td>CoolBox 96F System with CoolSink 96F</td><td>Essen Bioscience</td><td>1500-0078-A00</td><td></td></tr><tr><td>CoolBox with M30 System</td><td>Essen Bioscience</td><td>1500-0078-A00</td><td></td></tr><tr><td>Boyden Insert</td><td>Dominique Dutcher</td><td>353097</td><td></td></tr><tr><td>Boyden Coated Insert</td><td>Dominique Dutcher</td><td>354483</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Companion 24-well Plate</td><td>Dominique Dutcher</td><td>353504</td><td></td></tr><tr><td>BD Matrigel standard</td><td>BD BioScience</td><td>BD 354234</td><td>Store at -20&amp;deg;C.&amp;nbsp;</td></tr><tr><td>RAL 555 Staining Kit</td><td>RAL Diagnostics</td><td>&amp;nbsp;361550</td><td>Store in safe and dry place</td></tr><tr><td>Microcentrifuge tubes</td><td>Eppendorf</td><td>33511</td><td></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.
Figure 2 shows the Boyden membrane after cell fixation. Figure 2A shows a suboptimal result, with cell clusters on the upper side of the membrane. Figure 2B shows an optimal result with cells fixed and stained in blue on the membrane.
Figure 3 shows an optimal result of the scratch wound assay. The linear wound can be seen in Figure 3A. No cells are observed in the wound, and wound healing has occurred within 30 h. The time to heal the wound is dependent on the cell line and ranges from 24 to 50 h.
Figure 3B is a graphical representation of wound healing under four treatment conditions: control, ABT-199 (anti-Bcl-2), cetuximab (anti-EGFR), and ABT-199+cetuximab. The ABT-199+cetuximab combination significantly decreased cell migration. The curves are obtained using the video microscope software, which provides robust data analysis of cell migration with time. Error bars represent the standard deviation (SD) for each well.
Ninety percent confluence is the optimal cell density for the wound-healing assay. The optimal cell seeding depends on the cell line and ranges from 3 x 104 to 6 x 104 cells. Figure 4A shows the optimal cell density, and Figure 4C shows low cell density. Wells should be washed twice to avoid cell clusters, as shown in Figure 4B.
<img alt="Chemotaxis and invasion diagrams; porous membrane; cell migration; growth factors in assays." class="xfigimg" src="/files/ftp_upload/56337/56337fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Schematic representation of the Boyden chamber experiment. Inserts are placed into a companion plate with chemoattractant medium, and the cells are seeded in CM. The membrane can be uncoated (A) or coated (B).
<img alt="Microscope image comparing suboptimal (A) and optimal (B) experimental results; cellular distribution." class="xfigimg" src="/files/ftp_upload/56337/56337fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Suboptimal and optimal microscopic results obtained in the Boyden chamber experiment. (A) Suboptimal membrane with cell clusters on the upper side of the membrane and uninterpretable results. Scale bar = 300 µm. (B) Optimal membrane with countable cells in the lower part of the membrane stained in blue. Scale bar = 300 µm.
<img alt="Cell migration assay images and growth curve chart with drug treatment effects over 30 hours." class="xfigimg" src="/files/ftp_upload/56337/56337fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Optimal results obtained in the wound-healing experiment. (A) Representative image from the scratch wound assay shown wound healing observed at 0, 15, and 30 h. The criteria for a quality experiment are a linear wound, no cell fragments observed in the wound, and optimal cell confluence. Scale bar = 300 µm. (B) Graphical representation of wound healing showing quantification of parameters of wound cell confluence (percentage) according to time (h). Four treatment conditions are analyzed here, and the data are shown with the SD.
<img alt="Cell density comparison; microscopy image; ideal, washing issue, and low density; experimental results." class="xfigimg" src="/files/ftp_upload/56337/56337fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Suboptimal results obtained in the wound-healing experiment. (A) Ideal cell density. (B) Problems washing the cell pellet. (C) Low cell density. Scale bars = 300 µm
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Advantages</td>
			<td>Disadvantages</td>
		</tr>
		<tr>
			<td>Boyden Chamber Experiment</td>
			<td>-        3D-Cell Chemotaxis</td>
			<td>-        Time-Consuming</td>
		</tr>
		<tr>
			<td></td>
			<td>-        Both invasion and migration with coated and non coated layer matrix</td>
			<td>-        Low reproducibility</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>-        Expensive inserts</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>-        No Time-Lapse exploration</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>-        Need of adherent cells</td>
		</tr>
		<tr>
			<td>Wound Healing Assay</td>
			<td>-        Automatic Highly reproducible wound</td>
			<td>-        2D-Cell motility</td>
		</tr>
		<tr>
			<td></td>
			<td>-        Both invasion and migration with coated and non coated layer matrix</td>
			<td>-        Need of adherent cells</td>
		</tr>
		<tr>
			<td></td>
			<td>-        Cell proliferation included in the analysis</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>-        Time-Lapse microscopic visual analysis</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>-        Data exportation of wound healing metrics with precise analysis</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>-        Post-treatment robust software for cell migration and invasion curves</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Advantages and disadvantages of the Boyden chamber experiment and video microscope wound-healing assay. The advantages and disadvantages of the Boyden chamber experiment and scratch wound assay.

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 ...

evaluation-cell-invasion-migration-process-comparison-video

Nanyang Technological University

Research

JoVE Journal

Cancer Research

17.6K Views.  Université Lyon 1. Deze studie meldt twee verschillende methoden voor de analyse van de invasie van de cel en migratie: de bepaling van Boyden kamer en de in vitro video Microscoop gebaseerde wondgenezing bepaling. De protocollen voor deze twee experimenten worden beschreven, en hun voor- en nadelen worden vergeleken.

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

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.

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 ...

Developmental Biology

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. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

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

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

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

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 rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

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

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 significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

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

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

Medicine

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

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

A Customizable Chamber for Measuring Cell Migration

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

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

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

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for ...

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.

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

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