Name: high throughput traction force microscopy using pdms reveals dose-dependent effects of transforming growth factor-&#946; on the epithelial-to-mesenchymal transition
Uploaded: 2019-06-01T13:00:00
Description: Watch this Scientific Journal Video about High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-Î² on the Epithelial-to-Mesenchymal Transi

The authors thank Tom Kodger, Michael Landry, and Christopher J. Barrett for assistance with bead synthesis. A.J.E. acknowledges Natural Sciences and Engineering Research Council grants RGPIN/05843-2014 and EQPEQ/472339-2015, Canadian Institutes of Health Research grant no. 143327, Canadian Cancer Society grant no. 703930, and Canadian Foundation for Innovation Project #32749. R. Krishnan acknowledges National Institutes of Health grant no. R21HL123522 and R01HL136209. H.Y. was supported by Fonds de recherche Sant&#233; Qu&#233;bec, and Fonds de recherche Nature et Technologies Qu&#233;bec. The authors thank Johanan Idicula for assistance with the video and manuscript and Zixin He for assistance in preparing the video.

NOTE: The following protocol will guide researchers in fabricating and using the multi-well TFM dish shown in Figure 1.
1. Preparation of PDMS silicone substrates
<ol>
	<li>Preparation of PDMS silicone rubber mixture based on a composite mixture of two commercially available kits.
	<ol>
		<li>Add Part A and Part B of PDMS kit (e.g., GEL-8100, see the Table of Materials) in a 1:1 weight ratio into the 50 mL tube. 
		NOTE: The mixture is mixe...

<ol>
	<li>Harris, A., Wild, P., Stopak, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silicone+rubber+substrata:+a+new+wrinkle+in+the+study+of+cell+locomotion.">Silicone rubber substrata: a new wrinkle in the study of cell locomotion.</a> Science. 208 (4440), 177-179 (1980).</li><li>Oliver, T., Dembo, M., 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=Traction+forces+in+locomoting+cells.">Traction forces in locomoting cells.</a> Cell Motil Cytoskeleton. 31 (3), 225-240 (1995).</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 (4), 2307-2316 (1999).</li><li>Yoshie, H., Koushki, N., 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=Traction+Force+Screening+Enabled+by+Compliant+PDMS+Elastomers.">Traction Force Screening Enabled by Compliant PDMS Elastomers.</a> Biophysical Journal. 114 (9), 2194-2199 (2018).</li><li>Park, C. Y., Zhou, E. 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=High-throughput+screening+for+modulators+of+cellular+contractile+force.">High-throughput screening for modulators of cellular contractile force.</a> Integrative biology quantitative biosciences from nano to macro. 7 (10), 1318-1324 (2015).</li><li>Kraning-Rush, C. M., Califano, J. P., Reinhart-King, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+traction+stresses+increase+with+increasing+metastatic+potential.">Cellular traction stresses increase with increasing metastatic potential.</a> PLoS ONE. 7 (2), e32572 (2012).</li><li>Agus, D. B., 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=A+physical+sciences+network+characterization+of+non-tumorigenic+and+metastatic+cells.">A physical sciences network characterization of non-tumorigenic and metastatic cells.</a> Scientific Reports. 3 (1), 1449 (2013).</li><li>Guo, M., Ehrlicher, 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=Probing+the+Stochastic,+Motor-Driven+Properties+of+the+Cytoplasm+Using+Force+Spectrum+Microscopy.">Probing the Stochastic, Motor-Driven Properties of the Cytoplasm Using Force Spectrum Microscopy.</a> Cell. 158 (4), 822-832 (2014).</li><li>Ngan, E., Northey, J. J., Brown, C. M., Ursini-Siegel, J., Siegel, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+complex+containing+LPP+and+alpha-actinin+mediates+TGFbeta-induced+migration+and+invasion+of+ErbB2-expressing+breast+cancer+cells.">A complex containing LPP and alpha-actinin mediates TGFbeta-induced migration and invasion of ErbB2-expressing breast cancer cells.</a> Journal of Cell Science. 126 (Pt 9), 1981-1991 (2013).</li><li>Klein, S. M., Manoharan, V. N., Pine, D. J., Lange, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+monodisperse+PMMA+microspheres+in+nonpolar+solvents+by+dispersion+polymerization+with+a+macromonomeric+stabilizer.">Preparation of monodisperse PMMA microspheres in nonpolar solvents by dispersion polymerization with a macromonomeric stabilizer.</a> Colloid &amp; Polymer Science. 282 (1), 7-13 (2003).</li></ol>

For the success of this method, it is critical to have a uniformly coated sample with a constant thickness of approximately 100 &#181;m. The modulus should be carefully chosen to examine the physical significance of the biological system of interest. When fabricating a top layer, the concentration of the fiducial fluorescent particles should be optimized for accurate analysis of displacement and traction stress. Analyzing isolated single cells requires a denser fiduciary layer than measuring confluent monolayers. Additio...

Acto-myosin contractility is an essential element of active cell mechanics, impacting cell behaviors from motility and proliferation to stem cell differentiation. In tissues, contractility drives activity from polar separation in embryogenesis, to airway constriction and cardiac activity. Critically, to generate tension, cells must first adhere to their extracellular environment. In doing so, this contractility generates traction forces on their surroundings. Traction Force Microscopy (TFM) has emerged in a multitude of forms as a way to quantify these forces from diverse cells under different conditions.
The field of TFM has seen an exceptional breadth of innovation and application, and the results have paved the way for new perspectives in biology, which incorporate mechanics and physical forces. Starting with wrinkling silicone substrates1, researchers have applied various techniques to measure cell traction forces. These approaches have been continuously improved and have now reached a level of resolution on the order of several microns2. However, one principal problem has emerged, which is the difficulty in creating substrates of suitably low moduli using the available silicones. To circumvent this problem, polyacrylamide was adopted as a replacement due to the ease of creating substrates on the order of 1-20 kPa3. We recently implemented very compliant silicones in TFM4, allowing us to fabricate the same range of moduli as polyacrylamide, but with the advantages of inert and robust silicone.
TFM approaches have enabled valuable mechano-biological discoveries, however, a persistent shortcoming is their complexity, often restricting their use to researchers in the engineering or physical sciences disciplines. This is due&#160;in large part&#160;to the detailed calibrations and challenging calculations that are required to quantify contractility. Another significant challenge is that TFM methods are largely low-throughput and therefore ill-suited to study many different conditions or populations simultaneously5. This has presented a bottleneck, which has hampered transfer of TFM from a specialist biophysics setting into broader biological and pharmacological&#160;applications.
We have recently developed a multi-well format TFM plate, which allows researchers to parallelize their TFM measurements for faster quantification of contractility metrics, while exploring the impact of different compounds and also using less reagents4. This methodology has broad utility in diverse mechanobiology studies, from evaluating the effects of compounds on cellular activity, to quantifying the contractile changes in differentiation or disease.
One area of biomedical research that will benefit greatly from TFM is the study of how physical cues impact the malignant phenotypes of cancer cells. Metastasis, responsible for 90% of cancer-related deaths, is characterized by cancerous cells leaving their original tumor site and colonizing a secondary site. For cells to migrate through tissue and pass in and out of the vascular system, they must radically change their shapes to squeeze through these physical barriers while generating substantial forces to pull their way along extracellular matrix or move between other cells. These forces are transmitted to the substrate through focal adhesion interactions2,3, and can be quantified using TFM. While cancers are biochemically exceptionally diverse, with an expanding repertoire of known mutations and protein changes, some common physical changes have been observed; in a variety of cancers, including breast, prostate, and lung cancers, metastatic cells have been shown to exert 2-3 times the traction forces of non-metastatic cells6,7,8. These results suggest that there may be a strong correlation between metastatic progression and the traction forces exerted by cells; however, the detailed time-dependent changes in contractility are difficult to examine.
The epithelial-to-mesenchymal transition (EMT) is a process whereby cells reduce adherens- and tight-junction mediated cell-cell adhesion, becoming more migratory and invasive. In addition to physiological functions that include wound healing and developmental processes, EMT is also a process exploited during metastasis, making it a useful model system to study this process. Using TGF-&#946;, we can induce the EMT in&#160;murine mammary epithelial cells (NMuMG)9 to directly quantify the physical changes during this transformation, and characterize the time and dose-dependent effects of TGF-&#946; on EMT and cell contractility. In this article, we demonstrate the utility of this approach by measuring the changes in contractility during an induced EMT.

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		<tr height="42" style="height:31.2pt">
			<td class="xl69" height="42" style="height:31.2pt;width:206pt" width="275">GEL-8100</td>
			<td class="xl69" style="width:206pt" width="275">Nusil Technology</td>
			<td class="xl69" style="width:206pt" width="275">GEL-8100</td>
			<td class="xl70" style="width:206pt" width="275">High Purity Dielectric, Soft Silicone Gel kit</td>
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			<td class="xl70" height="42" style="height:31.2pt;width:206pt" width="275">Dow Corning Sylgard 184 Silicone Encapsulant Clear 0.5 kg Kit</td>
			<td class="xl69" style="width:206pt" width="275">Ellsworth Adhesives</td>
			<td class="xl69" style="width:206pt" width="275">184 SIL ELAST KIT 0.5KG</td>
			<td class="xl69" style="width:206pt" width="275">curing agent</td>
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			<td class="xl69" style="width:206pt" width="275">109.6mm&#177; x 72.8mm&#177; x 1mm thickness</td>
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			<td class="xl69" height="21" style="height:15.6pt;width:206pt" width="275">Target 2TM Nylon Syringe Filter</td>
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			<td class="xl69" height="21" style="height:15.6pt;width:206pt" width="275">96-well Stripwell Egg Crate Strip Holder</td>
			<td class="xl69" style="width:206pt" width="275">Corning</td>
			<td class="xl69" style="width:206pt" width="275">2572</td>
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			<td class="xl69" height="42" style="height:31.2pt;width:206pt" width="275">Polystyrene Universal Microplate Lid With Corner Notch&#160;</td>
			<td class="xl69" style="width:206pt" width="275">Corning</td>
			<td class="xl69" style="width:206pt" width="275">3099</td>
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			<td class="xl69" style="width:206pt" width="275">P016EA95</td>
			<td class="xl69" style="width:206pt" width="275">0.95</td>
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			<td class="xl69" height="21" style="height:15.6pt;width:206pt" width="275">2-Propanol</td>
			<td class="xl69" style="width:206pt" width="275">Sigma-Aldrich</td>
			<td class="xl69" style="width:206pt" width="275">190764</td>
			<td class="xl69" style="width:206pt" width="275">ACS reagent, &#8805;99.5%</td>
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			<td class="xl66" height="42" style="height:31.2pt;width:206pt" width="275">Fibronectin bovine plasma</td>
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			<td class="xl65" style="width:206pt" width="275">F1141-1MG</td>
			<td class="xl65" style="width:206pt" width="275">solution, sterile-filtered, BioReagent, suitable for cell culture</td>
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			<td class="xl64" height="21" style="height:15.6pt;width:206pt" width="275">DMSO</td>
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			<td align="right" class="xl64" style="width:206pt" width="275">472301</td>
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			<td class="xl77" colspan="4" height="21" style="height:15.6pt;
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			<td class="xl65" style="width:206pt" width="275">319-005-CL</td>
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			<td class="xl65" style="width:206pt" width="275">1M, free acid</td>
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			<td class="xl65" style="width:206pt" width="275">450-201-EL</td>
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			<td class="xl65" style="width:206pt" width="275">609-065-EL</td>
			<td class="xl65" style="width:206pt" width="275">200mM solution, sterile filtered for cell culture</td>
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			<td class="xl65" height="42" style="height:31.2pt;width:206pt" width="275">Amphotericine B</td>
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			<td class="xl65" style="width:206pt" width="275">450-105-QL</td>
			<td class="xl66" style="width:206pt" width="275">250&#956;g/ml, sterile filtered for cell culture</td>
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			<td class="xl66" height="21" style="height:15.6pt;width:206pt" width="275">Recombinant Human TGF-&#946;1</td>
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			<td class="xl65" style="width:206pt" width="275">100-21</td>
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			<td class="xl65" height="21" style="height:15.6pt;width:206pt" width="275">Acetic acid</td>
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			<td align="right" class="xl65" style="width:206pt" width="275">537020</td>
			<td class="xl65" style="width:206pt" width="275">Glacial, &#8805;99.85%</td>
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			<td class="xl65" style="width:206pt" width="275">251275&#160;</td>
			<td class="xl65" style="width:206pt" width="275">ACS reagent, &#8805;99.5%</td>
		</tr>
		<tr height="21" style="height:15.6pt">
			<td class="xl64" height="21" style="height:15.6pt;width:206pt" width="275">NMuMG</td>
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			<td class="xl64" style="width:206pt" width="275">Mouse Mammary Gland Cell Line</td>
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			<td class="xl64" style="width:206pt" width="275">AC190385000</td>
			<td class="xl64" style="width:206pt" width="275">99%, extra pure, ACROS Organics</td>
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			<td class="xl64" height="21" style="height:15.6pt;width:206pt" width="275">Potassium hydroxide</td>
			<td class="xl64" style="width:206pt" width="275">Sigma-Aldrich</td>
			<td align="right" class="xl64" style="width:206pt" width="275">221473</td>
			<td class="xl65" style="width:206pt" width="275">ACS reagent, &#8805;85%, pellets</td>
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		<tr height="21" style="height:15.6pt">
			<td class="xl64" height="21" style="height:15.6pt;width:206pt" width="275">TritonX-100</td>
			<td class="xl64" style="width:206pt" width="275">Sigma-Aldrich</td>
			<td class="xl64" style="width:206pt" width="275">X100</td>
			<td class="xl64" style="width:206pt" width="275">laboratory grade</td>
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			<td class="xl76" colspan="4" height="21" style="height:15.6pt;
 width:824pt" width="1100">Bead Synthesis</td>
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			<td class="xl67" height="62" style="height:46.8pt;width:206pt" width="275">1,1&#8242;-Dioctadecyl-3,3,3&#8242;,3&#8242;-tetramethylindocarbocyanine perchlorate (DiI)</td>
			<td class="xl67" style="width:206pt" width="275">Sigma-Aldrich&#160;</td>
			<td class="xl65" style="width:206pt" width="275">468495-100MG</td>
			<td align="right" class="xl72" style="width:206pt" width="275">97%</td>
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			<td class="xl67" height="42" style="height:31.2pt;width:206pt" width="275">Methyl methacrylate</td>
			<td class="xl67" style="width:206pt" width="275">Sigma-Aldrich&#160;</td>
			<td class="xl65" style="width:206pt" width="275">M55909-500ML</td>
			<td class="xl65" style="width:206pt" width="275">contains &#8804;30&#160;ppm MEHQ as inhibitor, 99%</td>
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			<td class="xl67" height="62" style="height:46.8pt;width:206pt" width="275">Inhibitor Remover</td>
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			<td class="xl68" height="42" style="height:31.2pt;width:206pt" width="275">Methacryloxylpropyl Terminated Polydimethylsiloxane</td>
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			<td class="xl67" style="width:206pt" width="275">Sigma-Aldrich&#160;</td>
			<td class="xl65" style="width:206pt" width="275">441090-25G</td>
			<td align="right" class="xl72" style="width:206pt" width="275">98%</td>
		</tr>
		<tr height="21" style="height:15.6pt">
			<td class="xl67" height="21" style="height:15.6pt;width:206pt" width="275">Hexane&#160;</td>
			<td class="xl67" style="width:206pt" width="275">Sigma-Aldrich&#160;</td>
			<td class="xl65" style="width:206pt" width="275">296090-2L</td>
			<td class="xl65" style="width:206pt" width="275">anhydrous, 95%</td>
		</tr>
		<tr height="21" style="height:15.6pt">
			<td class="xl67" height="21" style="height:15.6pt;width:206pt" width="275">Hexane, mixture of isomers</td>
			<td class="xl67" style="width:206pt" width="275">Sigma-Aldrich&#160;</td>
			<td class="xl65" style="width:206pt" width="275">227064-1L</td>
			<td class="xl65" style="width:206pt" width="275">anhydrous, &#8805;99%</td>
		</tr>
		<tr height="42" style="height:31.2pt">
			<td class="xl66" height="42" style="height:31.2pt;width:206pt" width="275">Whatman&#160;qualitative filter paper, Grade 1</td>
			<td class="xl65" style="width:206pt" width="275">Sigma-Aldrich&#160;</td>
			<td class="xl65" style="width:206pt" width="275">WHA1001055</td>
			<td class="xl65" style="width:206pt" width="275">circles, diam. 55&#160;mm,</td>
		</tr>
		<tr height="21" style="height:15.6pt">
			<td class="xl76" colspan="4" height="21" style="height:15.6pt;
 width:824pt" width="1100">Equipment</td>
		</tr>
		<tr height="21" style="height:15.6pt">
			<td class="xl73" height="21" style="height:15.6pt;width:206pt" width="275">Laurell WS-650Mz-23NPPB</td>
			<td class="xl64" style="width:206pt" width="275">Laurell Technologies</td>
			<td class="xl64" style="width:206pt" width="275"></td>
			<td class="xl71" style="width:206pt" width="275"></td>
		</tr>
		<tr height="21" style="height:15.6pt">
			<td class="xl74" height="21" style="height:15.6pt;width:206pt" width="275">UVP Handheld UV Lamp Model UVGL-58</td>
			<td class="xl64" style="width:206pt" width="275">VWR</td>
			<td class="xl74" style="width:206pt" width="275">21474-622</td>
			<td class="xl71" style="width:206pt" width="275"></td>
		</tr>
		<tr height="21" style="height:15.6pt">
			<td class="xl64" height="21" style="height:15.6pt;width:206pt" width="275">Rheometer</td>
			<td class="xl64" style="width:206pt" width="275">Anton Paar</td>
			<td class="xl64" style="width:206pt" width="275">MCR 302 WESP</td>
			<td class="xl71" style="width:206pt" width="275"></td>
		</tr>
	</tbody>
</table>

high throughput traction force microscopy using pdms reveals dose-dependent effects of transforming growth factor-&#946; on the epithelial-to-mesenchymal transition

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications,&#160;however, require direct quantification of these forces.
We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems.
In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-&#946;, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-&#946; exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

Before addition of TGF-&#946;, a confluent monolayer of cells has a cobblestone like shape and is tightly packed. Upon TGF-&#946; treatment, cells become more elongated in morphology, enlarging the cell area and acquiring a more mesenchymal phenotype. Utilizing the multi-well device fabricated with soft PDMS elastomers, the physical properties of cells in a total 17 different conditions were studied. The cells were treated with four different TGF-&#946; concentrations (0.5, 1, 2, and 4 ng...

Watch this Scientific Journal Video about High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-Î² on the Epithelial-to-Mesenchymal Transi

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-&#946; dependent increase in contractility during the epithelial-to-mesenchymal transition.

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...

My lab is interested in mechanobiology, which examines how cells exert and are influenced by mechanical forces.One way we can quantify mechanobiology is measuring cell contractility.In adherent cells, contractility manifests in traction forces exerted on the cell substrate.We quantify these forces using a methodology generally referred to as traction force microscopy or TFM.And TFM's implemented in a broad variety of different methods, but a common aspect is that it is a rather serial and slow lab technique done typically in a single dish like this one here.This restricts us to evaluating one population or condition at a time.Throughout in cell culture was improved some time ago but creating multiwell dishes and we thought, wouldn't it be great it we could to TFM with the same increased throughput and the same efficiency of time and reagents?And this was our solution.So we created a 96 well traction force dish.And it looks very similar to a conventional 96 well tissue culture dish, however with this dish we can not only examine 96 different cell culture conditions, but also quantify their cell contractility.This presents a much higher throughput solution than single dish approaches, allowing us to examine diverse cell conditions and contractile responses.During cancer metastasis, cancer cells travel from their primary site to secondary sites, spreading the disease in our body.This metastatic behavior of cancer cells accounts for 90%of cancer-related death.One of the proposed mechanisms for epithelial cancer cells to acquire a more migratory and invasive phenotype is epithelial-mesenchymal transition, EMT.During EMT, they become more migratory and invasive, changing their physical behavior.To better understand these changes in physical behavior, we utilize our soft silicon substrate to measure changes in contractility of mouse mammary gland cells with EMT induction by TGF-In this study, we investigated how different concentrations and incubation time of TGF-affect cells'physical behavior.Polydimethylsiloxane is a very compliant rubber with the lowest Young's modulus of about 0.6 kilopascal.The stiffness of this material also can be tunable using different concentrations of crosslink agent including SYLGARD 184.To measure mechanical properties of PDMS we use a rheometer.After mixing part A and part B of the PDMS, in a one to one weight ratio and adding the curing agent with the specified weight percentage, load the prepared batch on the rheometer plate.Make sure to load enough of the sample to completely cover the spindle area.It's important to make sure the spindle touches the sample completely with no free space left at its edge.Any extra sample should be carefully trimmed and wiped from the plate.Measure the final storage modulus and time dependency of each sample's viscoelasticity by doing time and frequency sweeps at an oscillatory shear strain of 0.5%Newsha]This graph is one example of time seq test, having done to measure rheological behavior of polydimethylsiloxane with applied oscillatory shear strain of 0.5%and temperature of 100

high-throughput-traction-force-microscopy-using-pdms-reveals-dose

Research

JoVE Journal

Bioengineering

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1) 1,2. Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. 
The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation 3,such as Ni2+ or Mg2+, which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase 4 and a repair enzyme 5, bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes 1,2 and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.

A tapping mode atomic force microscope (AFM) method for the visualization of plasmid DNA, cytoplasmic proteins, and DNA-protein complexes is described. The method includes alternate approaches for preparing samples for AFM imaging following biochemical manipulation. DNA containing specific protein interacting regions are observed in near-physiologic buffer conditions.

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their microenvironment. While many approaches exist for measuring cell-induced material strain, here we provide a methodology for monitoring strain with sub-micron resolution in a reference-free manner. Using a two-photon activated photolithographic patterning process, we demonstrate how to generate mechanically and bio-actively tunable synthetic substrates containing embedded arrays of fluorescent fiducial markers to easily measure three-dimensional (3D) material deformation profiles in response to surface tractions. Using these substrates, cell tension profiles can be mapped using a single 3D image stack of a cell of interest. Our goal with this methodology is to make traction force microscopy a more accessible and easier to implement tool for researchers studying cellular mechanotransduction processes, especially newcomers to the field.

This protocol provides instructions for implementing multiphoton lithography to fabricate three-dimensional arrays of fluorescent fiducial markers embedded in poly(ethylene glycol)-based hydrogels for use as reference-free, traction force microscopy platforms. Using these instructions, measurement of 3D material strain and calculation of cellular tractions is simplified to promote high-throughput traction force measurements.

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.

Collective cell migration in development, wound healing, and cancer metastasis is often guided by the gradients of growth factors or signaling molecules. Described here is an experimental system combining traction microscopy with a microfluidic system and a demonstration of how to quantify the mechanics of collective migration under biochemical gradient.

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

Traction Force Microscopy to Study B Lymphocyte Activation

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements from an experimentally observed displacement field produced by a cell pulling on an elastic substrate. Here, we adapted TFM to investigate the spatial and temporal structure of the force field exerted by B cells when activated by antigen engagement of the B cell receptor. Gel rigidity, bead density, and protein functionalization must be optimized for the study of relatively small cells (~ 6 &#181;m) that interact with, and respond specifically to ligands for cell surface receptors.

Here we present a protocol used to perform traction force microscopy experiments on B cells. We describe the preparation of soft polyacrylamide gels and their functionalization, as well as data acquisition at the microscope and a summary of data analysis.

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements ...