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

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 ...

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 ...

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 ...

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, ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...