The authors have nothing to disclose.

1. Preparation of Glass Coverslips for PAAs
<ol>
	<li>Clean 12 mm coverslips with low lint wipes.</li>
	<li>Flame the 12 mm coverslips and the 14 mm coverslip in the microwell of each 35 mm glass bottom dish by passing them through a Bunsen burner flame using tweezers.</li>
	<li>Treat the microwells with 200 &#181;l of 0.1 N NaOH for 5 min at room temperature.</li>
	<li>Aspirate and air dry the microwells for 30 min.</li>
	<li>Treat the microwells with 50-100 &#181;l of 3-aminopropyltrimethoxysilane for 10 min at room temperature in the fume hood. This chemical reacts with plastic; therefore, use glass pipettes and do not fill the microwells completely to avoid contact with the dish plastic.</li>
	<li>Wash the microwells with ultrapure water for approximately 10 min until the 3-aminopropyltrimethoxysilane becomes clear.</li>
	<li>Rinse the microwells with ultrapure water twice using a squeeze bottle.</li>
	<li>Wash the microwells with 2 ml of ultrapure water at room temperature on a rocker set at a medium speed (~1 Hz) for 10 min.</li>
	<li>Aspirate and air dry the microwells for 30 min.</li>
	<li>Treat the microwells with 2 ml of 0.5% glutaraldehyde solution at room temperature on a rocker set at a medium speed (~1 Hz) for 30 min.</li>
	<li>Wash the microwells with 2 ml of ultrapure water at room temperature on a rocker set at medium speed (~1 Hz) for 10 min. Repeat two more times for a total of 30 min.</li>
	<li>Dry microwells at a steep angle (60&#186; or greater) for 30 min. 
	Note: Dishes can be stored for 2 months in a dessicator.</li>
</ol>
2. Preparation of PAAs for Invadopodia Assays
<ol>
	<li>For a 1 ml solution of the soft PAA (8% acrylamide and 0.05% BIS), combine 200 &#181;l of 40% acrylamide, 25 &#181;l of 2% BIS, and 574 &#181;l of ultrapure water (Table 1).</li>
	<li>For a 1 ml solution of the hard PAA (8% acrylamide and 0.35% BIS), combine 200 &#181;l of 40% acrylamide, 175 &#181;l of 2% BIS, and 409 &#181;l of ultrapure water (Table 1).</li>
	<li>For a 1 ml solution of the rigid PAA (12% acrylamide and 0.60% BIS), combine 300 &#181;l of 40% acrylamide, 300 &#181;l of 2% BIS, and 169 &#181;l of ultrapure water (Table 1).</li>
	<li>Degas the solutions for 15 min. Add 200, 215, and 230 &#181;l of 1 mg/ml human plasma fibronectin in ultrapure water to the soft, hard, and rigid PAA solutions, respectively. For all solutions, add 1 &#181;l of 10 mg/ml acrylic acid N-hydroxysuccinimide (NHS) ester, 5 &#181;l of a 100 mg/ml ammonium persulfate, and 2 &#181;l of N,N,N&#8242;,N&#8242;-tetramethylethylenediamine (TEMED; Table 1). Mix with gentle pipetting and avoid creating bubbles in solutions.</li>
</ol>
<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>&#160; 
			PAA 
			(1 ml)</td>
			<td>40% 
			AA 
			(&#956;l)</td>
			<td>2% 
			BIS 
			(&#956;l)</td>
			<td>Ultrapure 
			Water 
			(&#956;l)</td>
			<td>1 mg/ml 
			FN 
			(&#956;l)</td>
			<td style="padding-left: 4px; padding-right: 4px;">10 mg/ml 
			NHS Ester 
			(&#956;l)</td>
			<td style="padding-left: 4px; padding-right: 4px;">100 mg/ml 
			APS 
			(&#956;l)</td>
			<td>&#160; 
			TEMED 
			(&#956;l)</td>
			<td>Elastic 
			Modulus 
			(Pa)</td>
		</tr>
		<tr>
			<td>Soft</td>
			<td>200</td>
			<td>25</td>
			<td>574</td>
			<td>200</td>
			<td>1</td>
			<td>5</td>
			<td>2</td>
			<td>1023</td>
		</tr>
		<tr>
			<td>Hard</td>
			<td>200</td>
			<td>175</td>
			<td>409</td>
			<td>215</td>
			<td>1</td>
			<td>5</td>
			<td>2</td>
			<td>7307</td>
		</tr>
		<tr>
			<td>Rigid</td>
			<td>300</td>
			<td>300</td>
			<td>169</td>
			<td>230</td>
			<td>1</td>
			<td>5</td>
			<td>2</td>
			<td>22692</td>
		</tr>
	</tbody>
</table>
Table 1: Ingredient volumes and resulting elastic moduli for soft, hard, and rigid PAAs used in the invadopodia assays. AA = acrylamide, FN = fibronectin, APS = ammonium persulfate.
<ol start="5">
	<li>Pipette 8.48 &#181;l of the PAA solution on to the center of each microwell. 
	Note: This volume theoretically yields a PAA of 75 &#181;m in thickness.</li>
	<li>Gently lower the flamed side of the 12 mm coverslip on to the droplet in the microwell using tweezers.</li>
	<li>Allow the sandwiched PAA solution to polymerize for 15-30 min. Verify polymerization by checking the leftover solution.</li>
	<li>Add 2 ml of 1x PBS to each dish and remove the 12 mm coverslips with tweezers. 
	Note: 10x PBS is made in the laboratory and composed of 0.011 M KH2PO4, 1.54 M NaCl, and 0.056 M Na2HPO4.</li>
	<li>Wash the microwells with 2 ml of 1x PBS at room temperature for 5 min. Repeat two more times for a total of 15 min.</li>
</ol>
3. Preparation of PAAs for Traction Force Assays
<ol>
	<li>For a 1 ml solution of the soft PAA (8% acrylamide and 0.05% BIS), combine 200 &#181;l of 40% acrylamide, 25 &#181;l of 2% BIS, and 566 &#181;l of ultrapure water (Table 2).</li>
	<li>For a 1 ml solution of the hard PAA (8% acrylamide and 0.35% BIS), combine 200 &#181;l of 40% acrylamide, 175 &#181;l of 2% BIS, and 401 &#181;l of ultrapure water (Table 2).</li>
	<li>For a 1 ml solution of the rigid PAA (12% acrylamide and 0.60% BIS), combine 300 &#181;l of 40% acrylamide, 300 &#181;l of 2% BIS, and 161 &#181;l of ultrapure water (Table 2).</li>
	<li>Degas the solutions for 15 min. Add 200, 215, and 230 &#181;l of 1 mg/ml human plasma fibronectin to the soft, hard, and rigid PAA solutions, respectively. Sonicate 8 &#181;l of 200 nm red fluorescent microspheres (excitation/emission of 580/605 nm) for 30 sec. For each solution, add 8 &#181;l of 200 nm red fluorescent microspheres, 1 &#181;l of 10 mg/ml acrylic acid NHS ester, 5 &#181;l of a 100 mg/ml ammonium persulfate, and 2 &#181;l of TEMED (Table 2). Mix with gentle pipetting and avoid creating bubbles in solutions.</li>
</ol>
<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td style="width:40px">&#160; 
			PAA 
			(1 ml)</td>
			<td>40% 
			AA 
			(&#956;l)</td>
			<td>2% 
			BIS 
			(&#956;l)</td>
			<td style="padding-left: 4px; padding-right: 4px;">Ultrapure 
			Water 
			(&#956;l)</td>
			<td>1 mg/ml 
			FN 
			(&#956;l)</td>
			<td>&#160; 
			Micro-spheres 
			(&#956;l)</td>
			<td style="padding-left: 4px; padding-right: 4px;">10 mg/ml 
			NHS Ester 
			(&#956;l)</td>
			<td style="padding-left: 4px; padding-right: 4px;">100 mg/ml 
			APS 
			(&#956;l)</td>
			<td>&#160; 
			TEMED 
			(&#956;l)</td>
			<td>Elastic 
			Modulus 
			(Pa)</td>
		</tr>
		<tr>
			<td>Soft</td>
			<td>200</td>
			<td>25</td>
			<td>566</td>
			<td>200</td>
			<td>8</td>
			<td>1</td>
			<td>5</td>
			<td>2</td>
			<td>1023</td>
		</tr>
		<tr>
			<td>Hard</td>
			<td>200</td>
			<td>175</td>
			<td>401</td>
			<td>215</td>
			<td>8</td>
			<td>1</td>
			<td>5</td>
			<td>2</td>
			<td>7307</td>
		</tr>
		<tr>
			<td>Rigid</td>
			<td>300</td>
			<td>300</td>
			<td>161</td>
			<td>230</td>
			<td>8</td>
			<td>1</td>
			<td>5</td>
			<td>2</td>
			<td>22692</td>
		</tr>
	</tbody>
</table>
Table 2: Ingredient volumes and resulting elastic moduli for soft, hard, and rigid PAAs used in the traction force assays. AA = acrylamide, FN = fibronectin, APS = ammonium persulfate.
<ol start="5">
	<li>Pipette 8.48 &#181;l of the PAA solution on to the center of each microwell. 
	Note: This volume theoretically yields a PAA of 75 &#181;m in thickness.</li>
	<li>Gently lower the flamed side of the 12 mm coverslip on to the droplet in the microwell using tweezers.</li>
	<li>Allow the sandwiched PAA solution to polymerize for 15-30 min. Verify polymerization by checking leftover solution.</li>
	<li>Add 2 ml of 1x PBS to each dish and remove the 12 mm coverslips with tweezers.</li>
	<li>Wash the microwells with 2 ml of 1x PBS at room temperature for 5 min. Repeat two more times for a total of 15 min.</li>
</ol>
4. Preparation of ECM for Invadopodia Assays
<ol>
	<li>Heat the gelatin solution (1% gelatin and 1% sucrose) to 37 &#176;C.</li>
	<li>Treat the PAAs with 150 &#181;l of the gelatin solution for 1 min then carefully aspirate from the bottom of the microwells when holding the glass bottom dishes at a 45&#176; angle.</li>
	<li>Dry the remaining thin layer of gelatin on the PAAs in the microwells at a steep angle (60&#176; or greater) to optimize drying for 60 min.</li>
	<li>Treat the microwells with 2 ml of a chilled 0.5% glutaraldehyde solution on ice for 15 min followed by room temperature for 30 min.</li>
	<li>Wash the microwells with 2 ml of a 1x PBS for 5 min. Repeat two more times for a total of 15 min.</li>
	<li>Treat the microwells with 2 ml of a sodium borohydride solution for 1 min. Gently tap dishes on bench to remove bubbles that form on the surface of the gelatin.</li>
	<li>Aspirate and wash the microwells with 2 ml of a 1x PBS for 5 min. Repeat two more times for a total of 15 min.</li>
	<li>Dilute a FITC-labeled human plasma fibronectin solution to 50 &#181;g/ml with a 1x PBS and centrifuge at 175,000 x g at 4 &#176;C for 15 min. Either purchase commercially available labeled fibronectin or label it as follows:
	<ol>
		<li>Dissolve 5 mg of fibronectin in 10 ml of borate buffer (170 mM sodium metaborate tetrahydrate and 40 mM NaCl) and place in dialysis tubing.</li>
		<li>Label the fibronectin by placing the tubing in 200 ml of borate buffer containing 6 mg of dissolved FITC at room temperature on a magnetic stirrer set at the lowest setting for 1.5 hr in the dark.</li>
		<li>Dialyze with 500 ml of a 1x PBS on a magnetic stirrer set at the lowest setting for 3 days in a dark cold room (4 &#176;C) with two volume changes a day.</li>
		<li>Calculate FITC-labeled fibronectin concentration based on the optical density of diluted aliquots (1:200) at 280 nm and 493 nm as [OD280 &#8211; (0.36 x OD493)] / 1.4 times the dilution factor of 200 times 2 (which accounts for the glycerol concentrating the fibronectin in the next step) resulting in mg/ml units.</li>
		<li>Dialyze overnight in a 50% glycerol solution on a magnetic stirrer set at the lowest setting overnight in a dark cold room at 4 &#176;C.)</li>
	</ol>
	</li>
	<li>Treat the microwells with 150 &#181;l of the FITC-labeled fibronectin solution at room temperature for 60 min in the dark.</li>
	<li>Carefully aspirate the FITC-labeled fibronectin solution from the bottom of the microwells when holding the glass bottom dishes at a 45&#176; angle and then fill the glass bottom dishes with 70% ethanol solution at room temperature for 10 min in a dark cell culture hood for sterilization. Also fill glass bottom dish lids or wipe clean with low lint wipes soaked in 70% ethanol.</li>
	<li>Wash glass bottom dishes by filling them with 1x PBS for 5 min. Repeat two more times with 2 ml of 1x PBS for a total of 15 min.</li>
</ol>
5. Preparation and Imaging of Invadopodia Assays
<ol>
	<li>Add 25,000 cancer cells in 2 ml of invadopodia medium (1:1 DMEM and RPMI 1640 with 5% low-protein serum, 10% fetal bovine serum, and 20 ng/ml of freshly added epidermal growth factor) to the glass bottom dishes and incubate overnight.</li>
	<li>Aspirate and treat the microwells with 2 ml of a 3.7% paraformaldehyde solution at room temperature in the dark for 20 min to fix the cells.</li>
	<li>After two quick washes with 2 ml of 1x PBS, treat the microwells with 2 ml of a 0.1% Triton X-100 solution at room temperature in the dark for 5 min to permeabilize the cells.</li>
	<li>After one quick wash with 2 ml of 1x PBS, treat the microwells with 3% bovine serum albumin blocking solution at room temperature in the dark for 60 min to block the cells.</li>
	<li>Incubate the microwells with 150 &#181;l of mouse anti-cortactin antibody (1:750) in blocking solution at room temperature in the dark for 60 min.</li>
	<li>Wash microwells with 2 ml of 1x PBS for 5 min. Repeat two more times for a total of 15 min.</li>
	<li>Incubate the microwells with 150 &#181;l of goat anti-mouse 633 antibody (1:500) and 546 phalloidin (1:750) in blocking solution at room temperature in the dark for 60 min.</li>
	<li>Wash microwells with 2 ml of 1x PBS for 5 min. Repeat two more times for a total of 15 min.</li>
	<li>Add six drops of mounting medium to fill each microwell and cover with 22 x 22 coverslips.</li>
	<li>Image cortactin and actin to identify invadopodia using excitation and emission filters of 632/647 nm and 556/570 nm, respectively, using a high NA, 40X oil immersion lens on a wide-field inverted microscope. Similarly, image fibronectin to identify ECM degradation using an excitation and emission filter of 492/518 nm.</li>
</ol>
6. Preparation and Imaging of Traction Force Assays
<ol>
	<li>Add 15,000 cancer cells in 2 ml of invadopodia medium to the glass bottom dishes and incubate overnight.</li>
	<li>Aspirate medium in the glass bottom dishes and replace with 2 ml of L-15 medium, with the same supplements (5% low-protein serum, 10% fetal bovine serum, and 20 ng/ml of freshly added epidermal growth factor), pre-warmed to 37 &#176;C.</li>
	<li>Incubate the glass bottom dishes in an environmental chamber on an inverted microscope pre-equilibrated to 37 &#176;C with high humidity for 1 hr.</li>
	<li>Take four sets of images using a high NA, 40X lens on a wide-field inverted microscope with an automated stage to mark cellular positions:
	<ol>
		<li>Image cells on top of the PAAs using phase contrast (&#8220;phase&#8221; image).</li>
		<li>Image fluorescent microspheres using excitation and emission filters of 560/645 nm by focusing slightly under the cells until the first layer of microspheres at the top of the PAAs comes into focus (&#8220;stressed&#8221; image).</li>
		<li>In addition, focus to the bottom of the PAAs (using the microspheres as markers) and take an image (&#8220;bottom&#8221; image). Note: The purpose of this image is to record the z-position to get the actual PAA thickness at each position for traction force calculations as described in Representative Results.</li>
		<li>After these images have been acquired at multiple positions, gently mix in 220 &#181;l of 10% Triton-X solution to remove cells. Image microspheres at the top of the PAAs at each position as previously described (&#8220;null&#8221; image).</li>
	</ol>
	</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+traction+force+and+measurement+methods.">Cell traction force and measurement methods.</a> Biomechanics and modeling in mechanobiology. 6, 361-371 (2007).</li><li>Dembo, M., Oliver, T., Ishihara, A., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+traction+stresses+exerted+by+locomoting+cells+with+the+elastic+substratum+method.">Imaging the traction stresses exerted by locomoting cells with the elastic substratum method.</a> Biophysical journal. 70, 2008-2022 (1996).</li><li>Dembo, M., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stresses+at+the+cell-to-substrate+interface+during+locomotion+of+fibroblasts.">Stresses at the cell-to-substrate interface during locomotion of fibroblasts.</a> Biophysical journal. 76, 2307-2316 (1999).</li><li>Engler, A. J., Rehfeldt, F., Sen, S., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtissue+elasticity:+measurements+by+atomic+force+microscopy+and+its+influence+on+cell+differentiation.">Microtissue elasticity: measurements by atomic force microscopy and its influence on cell differentiation.</a> Methods Cell Biol. 83, 521-545 (2007).</li><li>Kandow, C. E., Georges, P. C., Janmey, P. A., Beningo, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyacrylamide+hydrogels+for+cell+mechanics:+steps+toward+optimization+and+alternative+uses.">Polyacrylamide hydrogels for cell mechanics: steps toward optimization and alternative uses.</a> Methods in cell biology. 83, 29-46 (2007).</li><li>Leach, J. B., Brown, X. Q., Jacot, J. G., Dimilla, P. A., Wong, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurite+outgrowth+and+branching+of+PC12+cells+on+very+soft+substrates+sharply+decreases+below+a+threshold+of+substrate+rigidity.">Neurite outgrowth and branching of PC12 cells on very soft substrates sharply decreases below a threshold of substrate rigidity.</a> J Neural Eng. 4, 26-34 (2007).</li><li>Zhou, J., Kim, H. Y., Wang, J. H., Davidson, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroscopic+stiffening+of+embryonic+tissues+via+microtubules,+RhoGEF+and+the+assembly+of+contractile+bundles+of+actomyosin.">Macroscopic stiffening of embryonic tissues via microtubules, RhoGEF and the assembly of contractile bundles of actomyosin.</a> Development. 137, 2785-2794 (2010).</li><li>Buxboim, A., Rajagopal, K., Brown, A. E., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+deeply+cells+feel:+methods+for+thin+gels.">How deeply cells feel: methods for thin gels.</a> J Phys Condens Matter. 22, 194116 (2010).</li></ol>

This work was supported by the National Cancer Institute of the National Institutes of Health under Award Number K25CA143412 (Parekh). We would like to acknowledge that additional support was provided by the Department of Otolaryngology. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

We present a method for fabricating PAAs that can be used as the basis for invadopodia and traction force assays to correlate invasive and contractile cellular behaviors. While PAAs have long been used to look at rigidity effects on cells and calculate traction forces18,24,27, this protocol is the first to develop parallel assays based on PAAs with the same rigidities to correlate invasive and contractile cellular behaviors in response to matrix mechanical properties. Properly activating the coverslips of the glass bottom dishes ensures that the PAAs will bind to them and not come off. While we and others have relied on reagents such as sulfo-SANPAH and acrylic acid NHS ester to bind gelatin to the surface of the PAAs in the past4,12,28, we found that these methods did not produce reliably uniform surface layers of fibronectin. Therefore, fibronectin was embedded and crosslinked throughout the PAAs using acrylic acid NHS ester as performed by other groups29,30. However, increasing concentrations of fibronectin were required in order to yield the same ligand density at the surfaces of the soft, hard, and rigid PAAs5. Additionally, incorporation of fibronectin (but not the microspheres) reduced the mechanical properties of the soft, hard, and rigid PAAs from 1,071, 9,299, and 28,283 Pa4 to 1,023, 7,307, and 22,692 Pa5, respectively. However, these reduced elastic moduli values still encompassed the range of normal and cancerous tissues23. Storage moduli of the PAAs can easily be measured by rheometry and converted to elastic moduli1,4.
While the protocol is straight forward, lowering and removing the 12 mm coverslips are the most difficult steps and require practice. The biggest challenge when lowering a coverslip is to ensure that the PAA solution spreads out evenly without any bubbles or splashing. Removing a coverslip requires a steady hand in order to not damage the 12 mm coverslip, the glass bottom 14 mm coverslip, or the PAA. We have tried hydrophobic coatings on the 12 mm coverslip to aid in removal but found that the PAAs are left with patterns in their surfaces. The use of fine tip and thin, paddle-style tweezers for lowering and removing the 12 mm coverslips, respectively, is highly recommended. In addition, the amount of light used to image the cells must be minimized since some cell types are sensitive to concentrated light on the microscope. Also, L-15 medium was used for the traction force assays since our environmental chamber on our microscope is not equipped for CO2. While the same supplements were used in the media for the invadopodia and traction force assays in an attempt to keep conditions the same, DMEM and RPMI 1640 could be used instead of L-15 if CO2 levels can be controlled.
While the volume of PAA solution was chosen to theoretically yield gels with a thickness of 75 &#181;m, their thicknesses typically vary between 30-60 &#181;m. However, this range is well above the value at which cells can sense the underlying rigidity of the coverslips of the glass bottom dishes31. In addition, the thickness of the ECM layer used to detect degradation (gelatin and FITC-labeled fibronectin overlaid on to the PAAs) has been previously reported as approximately 1 &#181;m in thickness12; therefore, this thin layer does not shield the cells from the rigidities of the PAAs. However, solid debris, cracks, and other deformities in either the PAAs and/or ECM layer can affect individual cellular invasive and contractile properties. These problems can be caused by unclean 12 mm coverslips that introduce debris into the hydrogels, excessive drying of the gels and/or gelatin, and incomplete aspiration of sodium borohydride which can leave bubbles that deform the ECM layer. Therefore, care must be taken when selecting cells for imaging to ensure that they are not unduly influenced by local irregularities in the surfaces of the samples.
Relatively high concentrations of fibronectin in both the PAAs (mixed in at 200-230 &#181;g/ml) and the ECM layer (overlaid on the gelatin at 50 &#181;g/ml) have been used; however, the actual concentrations on either surface have not been directly measured. While each surface appears to be saturated with fibronectin, it is unclear whether cells experience the same exact ligand densities between the two assays. For the traction force assays, 200 nm microspheres at a dilution of 1:125 have proven optimal for imaging. However, it is quite common to find other groups using microspheres of different diameters or dilutions. In this system, smaller microspheres displayed Brownian motion within the PAAs, while larger microspheres caused cracks and deformities. These observations are most likely due to the physical properties of the PAAs (i.e., pore size, degree of crosslinking, etc.) that result from the specific amounts of acrylamide and BIS that were used. The microsphere dilution provides excellent resolution for tracking displacements in the PAAs, particularly since cancer cells exert relatively small forces. For improved optical resolution, particularly if using even smaller microspheres, high NA water or oil immersion objectives can be used.
Overall, many of these experimental factors can be adjusted based on the user&#8217;s requirements such as PAAs with different mechanical properties, cancer cells with varying invasive and contractile properties, and microscopy systems with other imaging capabilities. The ratio of acrylamide:BIS can be varied to produce PAAs with similar or different elastic moduli and crosslinking27. The sensitivity of the traction force assays can be adjusted to account for different cellular force levels by changing the rigidity and/or microsphere dilution. Different fluorophores can also be chosen for immunofluorescence and fibronectin labeling based on microscope specifications. While FITC does bleach, it is quite bright making ECM degradation easily identifiable. However, fluorescence imaging of invadopodia and small microspheres typically requires high NA objectives for optimal resolution. In addition, both assays can be performed on glass coverslips in well plates. However, glass bottom dishes are much easier to prepare and use throughout the experimental process. In the future, combining these assays into one would allow for direct visualization of both ECM degradation and cellular force generation. However, several technical challenges would arise including live cell imaging for invadopodia and microbead displacements, increased cellular exposure to light, bleaching of the FITC fibronectin, and whether traction forces are completely transduced through the fluorescently labeled and cross-linked ECM layer to the PAA surfaces.

The rigidity of the tumor-associated ECM has been identified as a significant factor in driving malignant behavior by increasing actomyosin contractility1-3. While this effect has primarily been demonstrated with breast cancer cells, matrix rigidity has been found to alter invasive properties of cells derived from a variety of cancers4-8 suggesting that tumor rigidity may play a role in other type of cancers. To penetrate cross-linked tissues during invasive migration, cancer cells utilize actin-rich adhesive protrusions known as invadopodia that localize proteinases to focally degrade the ECM9. Invadopodia are considered a hallmark of invasive cells and have been implicated in tumor cell invasion and metastasis10,11. Previous work has shown that matrix rigidity can regulate invadopodia numbers and associated ECM degradation4,12 through myosin II activity and mechanosensitive proteins12. Given the correlation between tumor density and cancer aggressiveness13,14, these results suggest a mechanism by which cancer cells may respond to rigid tumor tissues to drive invasion and metastasis through actomyosin contractility.
In vitro ECM rigidity and in vivo tissue density have been shown to regulate invasive behavior of cancer cells1,15-17. While actomyosin contractility appears to be important in this process, current studies conflict as to whether metastatic capacity is correlated to increased or decreased contractile forces6,18-20. Furthermore, it remains unknown whether these forces directly mediate invadopodia activity21. We recently found that cancer cell contractile forces were dependent on matrix rigidity and were predictive of ECM degradation by invadopodia5. These results suggest that cellular forces may play an important role in cancer progression by mediating invadopodia activity in response to the mechanical properties of the tumor microenvironment.
In order to correlate invasive and contractile properties of cancer cells5, we modified a protocol for creating PAAs with different rigidities that was previously used to investigate rigidity-dependent invadopodia activity4,12,22. By chemically crosslinking human plasma fibronectin throughout the PAAs, these modified hydrogels can be used as the basis for both invadopodia and traction force assays to ensure that cells experienced the same rigidities in both experiments5. In the invadopodia assays, the fibronectin provides a natural binding domain for gelatin to link the overlaid ECM to the PAAs to detect matrix degradation. In the traction force assays, the fibronectin provides a ligand for direct cellular adhesion to detect microsphere displacements used to calculate cellular traction forces. This method results in what we have called soft, hard, and rigid PAAs that are bound to glass bottom dishes and have elastic moduli, E, of 1,023, 7,307, and 22,692 Pa5 which span the range of mechanical properties reported for normal and cancerous tissues23.

<table><tbody><tr><td>3-Aminopropyltrimethoxysilane</td><td>Sigma-Aldrich</td><td>281778</td><td></td></tr><tr><td>Acrylamide (40%)</td><td>Bio-Rad</td><td>161-0140</td><td></td></tr><tr><td>Acrylic acid NHS ester</td><td>Sigma-Aldrich</td><td>A8060</td><td>prepare fresh in fume hood 10 mg/ml in DMSO</td></tr><tr><td>Alexa Fluor 546 phalloidin</td><td>Life Technologies</td><td>A22283</td><td>can also use rhodamine</td></tr><tr><td>Ammonium persulfate</td><td>Bio-Rad</td><td>161-0700</td><td>prepare fresh 10% solution in 1x PBS</td></tr><tr><td>Aqua Poly/Mount</td><td>Polysciences</td><td>18606</td><td>use 6 drops to fill microwells</td></tr><tr><td>BIS (2%)</td><td>Bio-Rad</td><td>161-0142</td><td></td></tr><tr><td>Bovine serum albumin</td><td>RPI</td><td>A30075</td><td>make 3% for blocking solution in 1x PBS and store in 4 &amp;deg;C</td></tr><tr><td>Coverslips (12 mm)</td><td>Fisher Scientific</td><td>12-545-80</td><td></td></tr><tr><td>dialysis tubing</td><td>Sigma-Aldrich</td><td>D9777</td><td>pre-equilibrate in borate buffer for 15 - 30 min</td></tr><tr><td>DMEM</td><td>Cellgro</td><td>10-013-CV</td><td>use to make invadopodia medium</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D8418</td><td>use to make acrylic acid NHS ester solution</td></tr><tr><td>Epidermal growth factor</td><td>Life Technologies</td><td>PHG0311</td><td>use to make invadopodia medium</td></tr><tr><td>Ethanol</td><td>PHARMCO-AAPER</td><td>E200</td><td>dilute with ultrapure water to 70%</td></tr><tr><td>FBS</td><td>Thermo Scientific</td><td>SH30070.03</td><td>use to make invadopodia medium</td></tr><tr><td>FITC</td><td>Sigma-Aldrich</td><td>F7250</td><td>protect from light</td></tr><tr><td>Gelatin</td><td>Polysciences</td><td>00639</td><td>typically make 10 ml of 1% sucrose/1% gelatin solution in PBS and store at 4 &amp;deg;C (preheat PBS to dissolve gelatin easily)</td></tr><tr><td>Glass bottom dishes (35 mm coverslips)</td><td>MatTek</td><td>P35G-0-14-C</td><td>coverslips are uncoated</td></tr><tr><td>Glutaraldehyde (25%)</td><td>Polysciences</td><td>01909</td><td>dilute with 1x PBS to 0.5%</td></tr><tr><td>Goat anti-mouse Alexa Fluor 633 antibody</td><td>Life Technologies</td><td>A21050</td><td></td></tr><tr><td>Human plasma fibronectin</td><td>Life Technologies</td><td>33016-015</td><td>add 5 ml of ultrapure water to make 1 mg/ml; aliquot in volumes based on use to avoid excessive freezing and thawing cycles</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>EMD Millipore</td><td>PX-1565-1</td><td>use to make 10x PBS stock</td></tr><tr><td>Mouse anti-cortactin 4F11 antibody</td><td>EMD Millipore</td><td>05-180</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>EMD Millipore</td><td>SX-0720-1</td><td>use to make 10x PBS stock</td></tr><tr><td>NaCl</td><td>RPI</td><td>S23020</td><td>use to make 10x PBS stock and borate buffer</td></tr><tr><td>NaOH (1 N)</td><td>Sigma-Aldrich</td><td>S2770</td><td>dilute with ultrapure water to 0.1 N</td></tr><tr><td>Nu-Serum (low-protein serum)</td><td>BD Biosciences</td><td>355500</td><td>use to make invadopodia medium</td></tr><tr><td>Paraformaldehyde</td><td>Acros</td><td>416785000</td><td>typically make 10% stock in 1x PBS, prepare in fume hood, and add a few ml of strong NaOH to dissolve paraformaldehyde easily then bring back to pH 7.4 with strong HCl)</td></tr><tr><td>PBS (sterile)</td><td>Cellgro</td><td>21-040-CV</td><td>use for cell culture</td></tr><tr><td>RPMI 1640</td><td>Cellgro</td><td>10-040-CV</td><td>use to make invadopodia medium</td></tr><tr><td>Sodium borohydride</td><td>Sigma-Aldrich</td><td>452882</td><td>prepare fresh in fume hood 1 mg/ml in 1x PBS</td></tr><tr><td>Sodium metaborate tetrahydrate</td><td>Sigma-Aldrich</td><td>S0251</td><td>use to make borate buffer</td></tr><tr><td>Sucrose</td><td>RPI</td><td>S24060</td><td>typically make 10 ml of 1% sucrose/1% gelatin solution in PBS and store at 4 &amp;deg;C (preheat PBS to dissolve gelatin easily)</td></tr><tr><td>TEMED</td><td>Bio-Rad</td><td>161-0800</td><td></td></tr><tr><td>Triton X-100</td><td>Alfa Aesar</td><td>A16046</td><td>make 10% stock in 1x PBS and use as is for cell removal in traction force assay or dilute with 1x PBS for staining</td></tr></tbody></table>

polyacrylamide gels for invadopodia and traction force assays on cancer cells

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is facilitated by actin-rich protrusions called invadopodia that proteolytically degrade the extracellular matrix (ECM). Invadopodia activity has been shown to be dependent on ECM rigidity and cancer cell contractile forces suggesting that rigidity signals can regulate these subcellular structures through actomyosin contractility. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. While some variations between the two assays exist, the protocol presented here provides a method for creating PAAs that can be used in both assays and are easily adaptable to the user&#8217;s specific biological and technical needs.

In the invadopodia assay, invadopodia are typically identified by colocalization of markers like actin and cortactin at punctate structures within the cell body (Figure 1). Both actively degrading and non-degrading invadopodia can be counted and are differentiated by whether these structures are colocalized with black areas lacking fluorescent signal in the FITC-labeled fibronectin (Figure 1). Invadopodia are manually counted, and ECM degradation per cell is determined by manually thresholding these black areas within outlines of the cells.
In the traction force assay, four images are captured at each cellular location marked by stage position (Figure 2). First, &#8220;phase&#8221; and &#8220;stressed&#8221; images are taken of all of the cells of interest. A &#8220;bottom&#8221; image of the PAA is also taken at each position in order to calculate hydrogel thickness. After removing the cells, a &#8220;null&#8221; image is taken at each marked stage position. Deformations in the PAAs are calculated based on the change in microsphere positions between the &#8220;stressed&#8221; and &#8220;null&#8221; images. These displacements and the mechanical properties of the PAAs (E and Poisson&#8217;s ratio assumed as 0.5) are then used to calculate traction forces (Figure 2). Several different methods exist for calculating traction forces24 based on some formulation of the Boussinesq solution for an infinite elastic half space25. While the details of these analyses are beyond the scope of this text, we have licensed LIBTRC software from Micah Dembo at Boston University which uses a previously described method for calculating traction forces26. This particular computational method compensates for finite thicknesses in its calculations which requires the thickness of the PAAs at each position which is calculated based on the difference in the z-position of the &#8220;stressed&#8221; and &#8220;bottom&#8221; images. Many research groups have similar computer packages available or choose to write their own programs. In addition, other methods exist for calculating traction forces based on different mathematical approaches24.
<img alt="Figure 1" src="/files/ftp_upload/52343/52343fig1highres.jpg" width="700" /> 
Figure 1: The invadopodia assay can be used to identify invadopodia and associated ECM degradation. Example wide-field fluorescence images of invadopodia in a SCC-61 (head and neck squamous cell carcinoma) cell on a hard PAA with 1% gelatin and FITC-labeled fibronectin. Invadopodia are typically identified by colocalization of two markers such as actin and cortactin (red and blue in the overlay image, respectively). Invadopodia can be quantitated as both actively degrading (colocalized with black areas lacking FITC signal as denoted by yellow arrows) and non-degrading (denoted by white arrows). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52343/52343fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52343/52343fig2highres.jpg" width="700" /> 
Figure 2: The traction force assay can be used to determine cellular traction forces generated by the actin cytoskeleton by tracking the displacement of embedded microspheres in the PAAs. Example wide-field phase and fluorescence images of a SCC-61 cell on a hard PAA (&#8220;phase&#8221;), and the microspheres directly under the cell at the top surface of the PAA (&#8220;stressed&#8221;). An image of the bottom of the PAA can also be taken to later calculate its local thickness (&#8220;bottom&#8221;). After the cell is removed, an image is once again taken of the microspheres at the top surface of the PAA (&#8220;null&#8221;). Microsphere positions between the &#8220;stressed&#8221; and &#8220;null&#8221; images can be tracked to yield a &#8220;displacement field.&#8221; Microsphere displacements and PAA mechanical properties can then be used to calculate a &#8220;traction vector field.&#8221; <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52343/52343fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells at JoVE.com

Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Mechanical rigidity in the tumor microenvironment plays a crucial role in driving malignant behavior by increasing invadopodia activity and actomyosin contractility. Using polyacrylamide gels (PAAs), invadopodia and traction force assays can be utilized to study the invasive and contractile properties of cancer cells in response to matrix rigidity.

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is ...

polyacrylamide-gels-for-invadopodia-traction-force-assays-on-cancer

Nanyang Technological University

Research

JoVE Journal

Medicine

12.0K Views.  Vanderbilt University Medical Center.  Mechanische stijfheid in de tumor micro-omgeving speelt een cruciale rol in het besturen van kwaadaardige gedrag door het verhogen van invadopodia activiteit en actomyosine contractiliteit. Met polyacrylamidegels (PTA) kunnen invadopodia en trekkracht assays worden gebruikt om de invasieve en contractiele eigenschappen van kankercellen in reactie op stijfheid matrix. 

Video: Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

Bioengineering

Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their extracellular matrix (ECM) environment. Eukaryotic cells constantly sense their local microenvironment through surface mechanosensors to transduce physical changes of ECM into biochemical signals, and integrate these signals to achieve specific changes in gene expression. Interestingly, physicochemical and mechanical parameters of the ECM can couple with each other to regulate cell fate. Therefore, a key to understanding mechanotransduction is to decouple the relative contribution of ECM cues on cellular functions.
Here we present a detailed experimental protocol to rapidly and easily generate biologically relevant hydrogels for the independent tuning of mechanotransduction cues in vitro. We chemically modified polyacrylamide hydrogels (PAAm) to surmount their intrinsically non-adhesive properties by incorporating hydroxyl-functionalized acrylamide monomers during the polymerization. We obtained a novel PAAm hydrogel, called hydroxy-PAAm, which permits immobilization of any desired nature of ECM proteins. The combination of hydroxy-PAAm hydrogels with microcontact printing allows to independently control the morphology of single-cells, the matrix stiffness, the nature and the density of ECM proteins. We provide a simple and rapid method that can be set up in every biology lab to study in vitro cell mechanotransduction processes. We validate this novel two-dimensional platform by conducting experiments on endothelial cells that demonstrate a mechanical coupling between ECM stiffness and the nucleus.

We present a new polyacrylamide hydrogel, called hydroxy-PAAm, that allows a direct binding of ECM proteins with minimal cost or expertise. The combination of hydroxy-PAAm hydrogels with microcontact printing facilitates independent control of many cues of the natural cell microenvironment for studying cellular mechanostransduction.

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 &#8211; 100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn&#8217;t require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a ...

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types&#39; responses to matrix stiffness have been characterized, how adherent cells&#39; susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, ...

Immunology and Infection

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts 

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Biology

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of&#160;single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance.

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes ...

Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

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