1. Fabrication of Deformable Polyacrylamide Hydrogels with Embedded Fluorescent Microspheres
NOTE: Directions describe polymerization of a 25 kPa hydrogel that is 22 &#956;m in diameter and approximately 66 &#956;m thick. Each or all of these parameters can be modified and directions to do so can be found in Table 1 and in the notes17.
<ol>
	<li>Activation of glass-bottom dishes or coverslips
	<ol>
		<li>Prepare the b...

<ol>
	<li>Acerbi, I., 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=Human+breast+cancer+invasion+and+aggression+correlates+with+ECM+stiffening+and+immune+cell+infiltration.">Human breast cancer invasion and aggression correlates with ECM stiffening and immune cell infiltration.</a> Integrative Biology (Camb. 7 (10), 1120-1134 (2015).</li><li>Mekhdjian, A. H., 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=Integrin-mediated+traction+force+enhances+paxillin+molecular+associations+and+adhesion+dynamics+that+increase+the+invasiveness+of+tumor+cells+into+a+three-dimensional+extracellular+matrix.">Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix.</a> Molecular Biology of the Cell. 28 (11), 1467-1488 (2017).</li><li>Paszek, M. J., 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=Tensional+homeostasis+and+the+malignant+phenotype.">Tensional homeostasis and the malignant phenotype.</a> Cancer Cell. 8 (3), 241-254 (2005).</li><li>Lange, J. R., Fabry, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+and+tissue+mechanics+in+cell+migration.">Cell and tissue mechanics in cell migration.</a> Experimental Cell Research. 319 (16), 2418-2423 (2013).</li><li>Davidson, L. A., Oster, G. F., Keller, R. E., Koehl, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+mechanical+properties+of+the+blastula+wall+reveal+which+hypothesized+mechanisms+of+primary+invagination+are+physically+plausible+in+the+sea+urchin+Strongylocentrotus+purpuratus.">Measurements of mechanical properties of the blastula wall reveal which hypothesized mechanisms of primary invagination are physically plausible in the sea urchin Strongylocentrotus purpuratus.</a> Developmental Biology. 209 (2), 221-238 (1999).</li><li>Li, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+mechanical+factors+in+fate+decisions+of+stem+cells.">Role of mechanical factors in fate decisions of stem cells.</a> Regenerative Medicine. 6 (2), 229-240 (2011).</li><li>Handorf, A. M., Zhou, Y., Halanski, M. A., Li, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+stiffness+dictates+development,+homeostasis,+and+disease+progression.">Tissue stiffness dictates development, homeostasis, and disease progression.</a> Organogenesis. 11 (1), 1-15 (2015).</li><li>Lo, C. M., Wang, H. B., 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=Cell+movement+is+guided+by+the+rigidity+of+the+substrate.">Cell movement is guided by the rigidity of the substrate.</a> Biophysical Journal. 79 (1), 144-152 (2000).</li><li>Kuo, C. H., Xian, J., Brenton, J. D., Franze, K., Sivaniah, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+stiffness+gradient+substrates+for+studying+mechanotactic+cell+migration.">Complex stiffness gradient substrates for studying mechanotactic cell migration.</a> Advanced Materials. 24 (45), 6059-6064 (2012).</li><li>Breckenridge, M. T., Desai, R. A., Yang, M. T., Fu, J., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrates+with+engineered+step+changes+in+rigidity+induce+traction+force+polarity+and+durotaxis.">Substrates with engineered step changes in rigidity induce traction force polarity and durotaxis.</a> Cell and Molecular Bioengineering. 7 (1), 26-34 (2014).</li><li>Goreczny, G. J., Wormer, D. B., Turner, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simplified+System+for+Evaluating+Cell+Mechanosensing+and+Durotaxis+In+Vitro.">A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro.</a> Journal of Visualized Experiments. (102), e52949 (2015).</li><li>Hartman, C. D., Isenberg, B. C., Chua, S. G., 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=Vascular+smooth+muscle+cell+durotaxis+depends+on+extracellular+matrix+composition.">Vascular smooth muscle cell durotaxis depends on extracellular matrix composition.</a> Proceedings of the National Acadademy of Sciences of the United States of America. 113 (40), 11190-11195 (2016).</li><li>Vincent, L. G., Choi, Y. S., Alonso-Latorre, B., del Alamo, J. C., Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell+durotaxis+depends+on+substrate+stiffness+gradient+strength.">Mesenchymal stem cell durotaxis depends on substrate stiffness gradient strength.</a> Biotechnology Journal. 8 (4), 472-484 (2013).</li><li>Sunyer, R., Jin, A. J., Nossal, R., Sackett, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+hydrogels+with+steep+stiffness+gradients+for+studying+cell+mechanical+response.">Fabrication of hydrogels with steep stiffness gradients for studying cell mechanical response.</a> PLoS One. 7 (10), e46107 (2012).</li><li>Martinez, J. S., Lehaf, A. M., Schlenoff, J. B., Keller, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+durotaxis+on+polyelectrolyte+multilayers+with+photogenerated+gradients+of+modulus.">Cell durotaxis on polyelectrolyte multilayers with photogenerated gradients of modulus.</a> Biomacromolecules. 14 (5), 1311-1320 (2013).</li><li>Plotnikov, S. V., Pasapera, A. M., Sabass, B., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+fluctuations+within+focal+adhesions+mediate+ECM-rigidity+sensing+to+guide+directed+cell+migration.">Force fluctuations within focal adhesions mediate ECM-rigidity sensing to guide directed cell migration.</a> Cell. 151 (7), 1513-1527 (2012).</li><li>McKenzie, A. J., 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=The+mechanical+microenvironment+regulates+ovarian+cancer+cell+morphology,+migration,+and+spheroid+disaggregation.">The mechanical microenvironment regulates ovarian cancer cell morphology, migration, and spheroid disaggregation.</a> Scientific Reports. 8 (1), 7228 (2018).</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Grashoff, 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=Measuring+mechanical+tension+across+vinculin+reveals+regulation+of+focal+adhesion+dynamics.">Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics.</a> Nature. 466 (7303), 263-266 (2010).</li><li>McKenzie, A. J., Campbell, S. L., Howe, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+A+activity+and+anchoring+are+required+for+ovarian+cancer+cell+migration+and+invasion.">Protein kinase A activity and anchoring are required for ovarian cancer cell migration and invasion.</a> PLoS One. 6 (10), e26552 (2011).</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>Plotnikov, S. V., Sabass, B., Schwarz, U. S., Waterman, C. 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Demonstrated here is a repeatable, single-cell durotaxis assay that allows assessment of a cell&#8217;s ability to alter its migration behavior in response to acute mechanical cues. This technique can also be used in combination with fluorescence microscopy and appropriate fusion proteins or biosensors to examine subcellular signaling and cytoskeletal events within seconds of mechanical stimulation or over a longer timescale during durotactic movement. Understanding a cell&#8217;s relationship to its environment involves...

Over the past few decades, the importance of the mechanical properties of a cell&#8217;s environment has garnered increasing recognition in cell biology. Different tissues and extracellular matrices have different relative stiffnesses and, as cells migrate throughout the body, they navigate these changes, using these mechanical properties to guide them1,2,3,4,5,6,7. Cells use the stiffness of a given tissue to inform their motile behavior during processes such as development, wound healing, and cancer metastasis. However, the molecular mechanisms that allow sensation of and response to these mechanical inputs remain largely unknown1,2,3,4,5,6,7.
In order to study the mechanisms through which cells respond to physical environmental cues, the rigidity or stiffness of the substrate underlying adherent cells must be manipulated. In 2000, Chun-Min Lo, Yu-Li Wang and colleagues developed an assay8 whereby an individual cell&#8217;s motile response to changing mechanical cues could be directly tested by stretching deformable extracellular matrix (ECM)-coated polyacrylamide hydrogels on which the cells were plated. Cells exhibits a significant preference for migrating towards stiffer substrates, a phenomenon they dubbed &#8220;durotaxis.&#8221;
Since the original report in 2000, many other techniques have been employed for the study of durotaxis. Steep stiffness gradients have been fabricated by casting gels over rigid features such as polystyrene beads9 or stiff polymer posts10 or by polymerizing the substrate around the edges of a glass coverslips11 to create mechanical &#8216;step-boundaries&#8217;. Alternatively, hydrogels with shallower but fixed stiffness gradients have been fabricated by a variety of methods such as gradients of crosslinker created by microfluidic devices12,13 or side-by-side hydrogel solution droplets of differing stiffness8, or hydrogels with photoreactive crosslinker treated with graded UV light exposure to create a linear stiffness gradient14,15. These techniques have been used to great effect to investigate durotactic cellular movement en masse over time. However, typically these features are fabricated in advance of cell plating and their properties remain consistent over the course of the experiment, relying on random cell movement for sampling of mechanical gradients. None of these techniques are amenable to observation of rapid changes in cellular behavior in response to acute mechanical stimulus.
In order to observe cellular responses to acute changes in the mechanical environment, single cell durotaxis assays offer several advantages. In these assays, individual cells are given an acute, mechanical stimulus by pulling the underlying substrate away from the cell with a glass micropipette, thereby introducing a directional gradient of cell-matrix tension. Changes in the motile behavior, such as speed or direction of migration, are then observed by live-cell phase contrast microscopy. This approach facilitates direct observation of cause and effect relationships between mechanical stimuli and cell migration, as it allows rapid, iterative manipulation of the direction and magnitude of the tension gradient and assessment of consequent cellular responses in real time. Further, this method can also be used to mechanically stimulate cells expressing fluorescent fusion proteins or biosensors to visualize changes in the amount, activity, or subcellular localization of proteins suspected to be involved in mechanosensing and durotaxis.
This technique has been employed by groups who study durotaxis8,16 and is described here as it has been adapted by the Howe Laboratory to study the durotactic behavior of SKOV-3 ovarian cancer cells and the molecular mechanisms that underly durotaxis17. Additionally, a modified method is described for fabrication of hydrogels with a single, even layer of fluorescent microspheres near the cell culture surface; this facilitates visualization and optimization of micropipette-generated strain gradients and may allow assessment of cell contractility by traction force microscopy.

<table>
	<tbody>
		<tr>
			<td>Acrylamide 40 %&#160;</td>
			<td>National Diagnostic</td>
			<td>EC-810</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Persulfate&#160;</td>
			<td>Fisher</td>
			<td>BP179-25</td>
			<td></td>
		</tr>
		<tr>
			<td>BD20A High frequency generator</td>
			<td>Electro Technic Products</td>
			<td>12011A</td>
			<td>115 V -&#160;Handheld Corona Wand</td>
		</tr>
		<tr>
			<td>Bind Silane (y-methacryloxypropyltrimethoxysilane) (</td>
			<td>Sigma Aldrich</td>
			<td>M6514</td>
			<td></td>
		</tr>
		<tr>
			<td>Bis-acrylamide 2%&#160;</td>
			<td>National Diagnostic</td>
			<td>EC-820</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson 2510 Ultrasonic Cleaner</td>
			<td>Bransonic</td>
			<td></td>
			<td>40 kHz frequency</td>
		</tr>
		<tr>
			<td>Coarse Manipulator</td>
			<td>Narshige</td>
			<td>MC35A</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM without phenol red</td>
			<td>Sigma Aldrich</td>
			<td>D5030</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-Stage Glass Micropipette Puller</td>
			<td>Narshige</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Pharmco-aaper</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (Qualified One Shot)</td>
			<td>Gibco</td>
			<td>A31606-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin&#160;</td>
			<td>EMD Millipore</td>
			<td>FC010</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluospheres Carboxylate 0.2 um&#160;</td>
			<td>Invitrogen</td>
			<td>F8810, F8807, F8811</td>
			<td></td>
		</tr>
		<tr>
			<td>Fugene 6</td>
			<td>Roche</td>
			<td>1815091</td>
			<td>1.5 ug DNA / 6uL fugene 6 per 35mm dish</td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>Fisher Chemical</td>
			<td>A38SI-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Dish</td>
			<td>CellVis</td>
			<td>D60-60-1.5-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Coverslip</td>
			<td>Electron Microscopy Sciences</td>
			<td>72224-01</td>
			<td>22 mm, #1.5</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>JT Baker</td>
			<td>9535-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Hellmanex III&#160;Special cleaning concentrate</td>
			<td>Sigma Aldrich</td>
			<td>Z805939</td>
			<td>Used at 2% in ddH2O for cleaning coverslips</td>
		</tr>
		<tr>
			<td>HEPES powder</td>
			<td>Sigma Aldrich</td>
			<td>H3375</td>
			<td>Make 50mM HEPES buffer, pH 8.5</td>
		</tr>
		<tr>
			<td>Intelli-Ray 400 Shuttered UV Flood Light</td>
			<td>Uviton International</td>
			<td>UV0338</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Chemical</td>
			<td>A417-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narshige</td>
			<td>MF900</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Narshige</td>
			<td>MHW3</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Sigma Aldrich</td>
			<td>M5904</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanopure Life Science UV/UF System</td>
			<td>Barnstead</td>
			<td>D11931</td>
			<td>ddH2O</td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OptiMEM</td>
			<td>Invitrogen</td>
			<td>31985062</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Bemis Company, Inc</td>
			<td>PM-992</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td>139 mM NaCl, 2.5 mM KCl, 28.6 mM Na2HPO4, 1.6 mM KH2PO4, pH 7.4</td>
		</tr>
		<tr>
			<td>Platelet Derived Growth Factor-BB (PDGF-BB)</td>
			<td>Sigma Aldrich</td>
			<td>P4056</td>
			<td></td>
		</tr>
		<tr>
			<td>Ref52</td>
			<td></td>
			<td></td>
			<td>Rat embryonic fibroblast cell line; Culture in DMEM + 10% FBS</td>
		</tr>
		<tr>
			<td>Ringer&#39;s Buffer</td>
			<td></td>
			<td></td>
			<td>134 mM NaCl, 5.4 mM KCl, 1 mM MgSO4, 2.4 mM CaCl2, 20 mM HEPES, 5 mM D-Glucose, pH 7.4</td>
		</tr>
		<tr>
			<td>SKOV-3</td>
			<td>American Type Culture Collection</td>
			<td></td>
			<td>Culture in DMEM + 10% FBS</td>
		</tr>
		<tr>
			<td>Sulfo-SANPAH&#160;</td>
			<td>Covachem</td>
			<td>&#160;12414-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabletop Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-32G</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED&#160;</td>
			<td>Sigma Aldrich</td>
			<td>T9281-50</td>
			<td></td>
		</tr>
	</tbody>
</table>

single cell durotaxis assay for assessing mechanical control of cellular movement and related signaling events

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate underlying a cell must be manipulated. While hydrogels with graded stiffness and long-term migration assays have proven useful in durotaxis studies, immediate, acute responses to local changes in substrate tension allow focused study of individual cell movements and subcellular signaling events. To repeatably test the ability of cells to sense and respond to the underlying substrate stiffness, a modified method for application of acute gradients of increased tension to individual cells cultured on deformable hydrogels is used which allows for real time manipulation of the strength and direction of stiffness gradients imparted upon cells in question. Additionally, by fine tuning the details and parameters of the assay, such as the shape and dimensions of the micropipette or the relative position, placement, and direction of the applied gradient, the assay can be optimized for the study of any mechanically sensitive cell type and system. These parameters can be altered to reliably change the applied stimulus and expand the functionality and versatility of the assay. This method allows examination of both long term durotactic movement as well as more immediate changes in cellular signaling and morphological dynamics in response to changing stiffness.

By preparing micropipettes (Figure 1) and normalizing the force generation of the pulls (Figure 2 and Figure 3) as described above, optimal durotactic conditions have been identified for multiple cell lines. Using this technique, as outlined in Figure 4, both SKOV-3 ovarian cancer cells17&#160;and Ref52 rat embryonic fibroblasts (Figure 5) move towar...

Watch this Scientific Journal Video about Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events at JoVE.com

Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events

Mechanical forces are important for controlling cell migration. This protocol demonstrates the use of elastic hydrogels that can be deformed using a glass micropipette and a micromanipulator to stimulate cells with a local stiffness gradient to elicit changes in cell structure and migration.

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate ...

There's growing appreciation for the impact of the mechanical microenvironment on cell signaling and migration. Necessitating the use of unique, experimental approaches to deliver mechanical stimulation to individual cell. This technique allows the dynamic manipulation of the mechanical micro-environment underneath a cell in real time.Allowing the observation of mechanically regulated changes in cell migration behavior, and sub-cellular signaling events. Demonstrating the procedure with me will be Nyla Naim, a post-doc and Johnathan Patterson, a technician from our laboratory. Begin by using a Chorono-1 to activate the glass surface of each bottom cover slip for 20 seconds, before immediately over-laying 50 microliters of bind silane working solution onto each cover slip.Allow the solution to dry for 10 minutes before rinsing the cover slips two times with 95%ethanol and two times with isopropanol. Then, allow the cover slips to air dry for approximately 20 minutes. For fluorescent micro-bead deposition, first sonicate the working micro-bead solution for one hour.15 minutes before the end of the sonication, place the activated cover slips vertically in a ceramic cover slip holder and transfer the holder into a table-top plasma cleaner for room-air plasma treatment for three minutes. Next, place pieces of paraffin film in 60 milliliter Petri dish lids, and place the cover slips onto the films, lightly tapping each cover slip to ensure a good contact between the film and the coverslip. Then, add 150 microliters of the working bead solution to the top of each coverslip, and immediately aspirate the ethanol solution from the side of the coverslips, leaving the beads on the coverslip.To cast the Hydrogels immediately after their preparation, add a 25 microliter droplet of Hydrogel solution to the activated side of each bottom coverslip, and immediately flip the beaded top coverslips, bead-side down, onto the droplet. Allow the gels to polymerize for 30 minutes before using forceps to gently remove the coverslips. Then, rinse the gels with three, five minute washes in fresh 50 miliMoler Hepes per wash.To activate the Hydrogels surfaces, incubate the gels in fresh 50 miliMoler Hepes, supplemented with 0.4 miliMolar Sulfo Sanpah, and immediately expose the gels to an ultraviolet arch lamp in an enclosed area for 90 seconds. At the end of the activation, wash the gels with three, five minute washes in fresh Hepes, and treat the activated Hydrogels with 20 micrograms per milliliter of fibronectin for one hour at 37 degrees Celsius. At the end of the incubation, aspirate the excess fibronectin solution, and wash the gels three times in PBS for five minutes per wash.After the last wash, place the Hydrogels in a small volume of PBS and sterilize the gels for 15 minutes under ultra-violet light in a tissue culture hood. Then, wash the gels one time in fresh PBS. For cell plating, seed 2.1 times 10 the four of the cells of interest in three milliliters of medium per-Hydrogel and allow the cells to adhere at 37 degrees Celsius for four to 18 hours.While the cells are equilibrating, use a micro-pipette puller to pull a one millimeter diameter by 100 millimeter long borosilicate glass micro-pipette, to obtain a taper of over two millimeters that reduces to approximately 50 micrometers in diameter in the first millimeter, and extends to a long, parallel 10 micrometer diameter tube in the last millimeter. Next, load the pipette into a microforge and shape the pipette to have a 15 micrometer blunted tip that is enclosed at the very end of a 250 micrometer section, bent at approximately 35 degree angel from the rest of the pipette. The approximate diameter at the bend should be around 30 micrometers to lend strength to the tip.Next, place a Hydrogel onto the stage of an inverted microscope. Cover the medium with mineral oil and select the 10x objective. Sterilize the prepared micro-pipette with the 70%alcohol.Then, insert into the micro-pipette holder with the hook pointed toward the dish, and use the course manipulator to lower the pipette until the hook just touches the surface of the liquid. Focus the microscope on the cells adhered to the top of the gel, and taking care that the objective is in no danger of hitting the sample or stage, bring the focus above the gel to find the tip of the micro-pipette. Rotate the pipette until the tip is perpendicular to the focal plane, so that the blunted tip of the micro-pipette is pointing down, and refocus on the tip of the pipette as necessary.Focus back down to the cell layer to gauge how far the pipette is from the gel surface, and focus back up to a plane that is part-way between the gel and the tip of pipette. Then, slowly lower the pipette to reach the intermediate focal plane, and increase the magnification to that which will be used in the experiment, before lowering the pipette until it hovers just above the surface of the Hydrogel. For micro-manipulator calibration, image an area devoid of cells, an area with beads, but no manipulation, with the pipette engaging the gel, and with the engaged pipette pulling the gel.Then, use image J to compare the no bead fields to the bead fields without engagement, the bead fields with the gel engaged, and the pulled gel to calculate the relative bead displacements and force applied to the gels. To conduct a durotaxis assay, monitor a group of cells for 30 minutes to identify cells that are moving in a directed manner. Select a cell that is steadily moving in one direction, and monitor the cells of the desired frame-rate for an additional 30 minutes.Next, position the pipette about 50 micrometers in front of the near side of the leading edge of the selected cell, and move the micro-manipulator such that the gel is deformed orthogonally to the cell's direction of travel. Then, observe the cell over time as it responds to the acute, local gradient of Hydrogel stiffness. Using this technique as demonstrated, rat embryonic fibroblasts move towards the increased stiffness in gradients applied by a glass micro-pipette.Rat embryonic fibroblast cells, transiently expressing vinculin tension sensor on 125 kilopascal polyacrylamide gels also demonstrate the formation of focal adhesions in the directions of the stretch over a period of 40 minutes. Forster Resonance Energy Transfer analysis reveals that vinculin localized to focal adhesions experiences an immediate change in tension, when presented with an acute durotactic stimulation. Expanding the utility of this assay to the observation of sub-cellular signaling events, in response to durotactic stimulation.If you make multiple attempts to stretch a cell, or if the microneedle slips during the assay, start over with a new cell to avoid imparting multiple, variable, mechanical stimuli. Beyond its utility for assaying durotaxis, this technique can be combined with genetic approaches, such as biosensors, RNAI, or gene editing to help delineate the molecular mechanisms involved in mechanotransduction.

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JoVE Journal

Biology

7.7K Views.  University of Vermont Larner College of Medicine. There's growing appreciation for the impact of the mechanical microenvironment on cell signaling and migration. Necessitating the use of unique, experimental approaches to deliver mechanical stimulation to individual cell. This technique allows the dynamic manipulation of the mechanical micro-environment underneath a cell in real time.Allowing the observation of mechanically regulated changes in cell migration behavior, and sub-cellular signaling events. Demonstrating the procedure wit...