Name: stiffness measurement of soft silicone substrates for mechanobiology studies using a widefield fluorescence microscope
Uploaded: 2018-07-03T14:00:00
Description: Watch this Scientific Journal Video about Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope at JoVE.com

Agradecemos Margaret Gardel generosamente, permitindo o uso da rheometer. Reconhecemos o apoio do NIH (1R15GM116082) que permitiu a este trabalho.

1. fabricação de substrato de Silicone suave
<ol><li>Pesar 1,75 g de componente e 1,75 g do componente B (A:B = 1:1) do kit de elastômero de silicone suave usando (poliestireno) pesando bandejas.</li>
	<li>Adicionar o componente para o componente B na bandeja de pesagem e misturá-los juntos por 5 min utilizando uma ansa apropriado.</li>
	<li>Adicione a mistura acima para uma placa de Petri de 35mm. Deixe a mistura espalhar uniformemente em toda a placa de Petri por alguns minutos. Nota: A escolha do diâmetro do...

<ol>
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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, 144-152 (2000).</li><li>Discher, D. E., Janmey, P., 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=Tissue+cells+feel+and+respond+to+the+stiffness+of+their+Substrate.">Tissue cells feel and respond to the stiffness of their Substrate.</a> Science. 310, 1139-1143 (2005).</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>Johnston, I. D., McCluskey, D. K., Tan, C. K. L., Tracey, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+characterization+of+bulk+Sylgard+184+for+microfluidics+and+microengineering.">Mechanical characterization of bulk Sylgard 184 for microfluidics and microengineering.</a> Journal of Micromechanics and Microengineering. 24 (3), 035017 (2014).</li><li>Style, R. W., 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+microscopy+in+physics+and+biology.">Traction force microscopy in physics and biology.</a> Soft Matter. 10 (23), 4047-4055 (2014).</li><li>Lee, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+the+cytoplasmic+domain+of+N-cadherin+reduces,+but+does+not+eliminate,+traction+force-transmission.">Deletion of the cytoplasmic domain of N-cadherin reduces, but does not eliminate, traction force-transmission.</a> Biochemical and Biophysical Research Communications. 478 (4), 1640-1646 (2016).</li><li>Frey, M. T., Engler, A., Discher, D. E., Lee, J., 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=Microscopic+methods+for+measuring+the+elasticity+of+gel+substrates+for+cell+culture:+microspheres,+microindenters,+and+atomic+force+microscopy.">Microscopic methods for measuring the elasticity of gel substrates for cell culture: microspheres, microindenters, and atomic force microscopy.</a> Methods Cell Biol. 83, 47-65 (2007).</li><li>Lee, D., Rahman, M. M., Zhou, Y., Ryu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+confocal+microscopy+indentation+method+for+hydrogel+elasticity+measurement.">Three-dimensional confocal microscopy indentation method for hydrogel elasticity measurement.</a> Langmuir. 31 (35), 9684-9693 (2015).</li><li>Dimitriadis, E. K., Horkay, F., Maresca, J., Kachar, B., Chadwick, R. 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Enquanto o método de indentação de esfera é fácil de implementar, cuidado deve prestar atenção para a escolha de indentor e a espessura da amostra silicone macio. A equação utilizada para calcular o módulo de Young é válida sob um conjunto de condições11e estas normalmente são satisfeitas quando a espessura da amostra do silicone é > 10% do raio indentor e < ~ 13 x o raio indentor. Descobrimos que uma espessura do silicone de 5-10 x o raio indentor foi uma boa escolha, no qual a es...

Células em tecidos moles residem em um microambiente cuja rigidez é o hectopascal gama1, em contraste com pratos de cultura de tecidos cuja rigidez é várias ordens de magnitude maior. Primeiras experiências com células em substratos de macio revestido de proteínas da matriz extracelular mostraram que a rigidez de substrato influencia como células seguir em frente, bem como aderirem à matriz extracelular abaixo de2,3. Na verdade, a rigidez de substrato influencia fundamentalmente a função celular4 de maneira semelhante aos sinais bioquímicos penetrante. Gel de poliacrilamida (revestido com proteínas da matriz extracelular) são (água-permeando) hidrogel que têm sido amplamente utilizados como substratos de cultura de células para estudos de mechanobiology5. Polydimethylsiloxane (PDMS), o mais comum do silicone (silicone), foi amplamente utilizado como um silicone duro com rigidez megapascal-gama para mícron-escala de fabricação6. Mais recentemente, silicone macio substratos com rigidez na faixa hectopascal mais fisiologicamente relevantes têm sido empregados como substratos de cultura celular para mechanobiology estudos7,8.
Vários métodos têm sido utilizados para medir a dureza de substratos flexíveis, incluindo microscopia de força atômica, a deformação macroscópica das amostras toda sobre o alongamento, reologia e recuo usando esferas e Ponta esférica microindentors9 . Enquanto cada técnica tem suas próprias vantagens e desvantagens, recuo com uma esfera é um método especialmente simples, mas bastante preciso que requer apenas o acesso a um microscópio de fluorescência widefield. Recuo com uma esfera metálica tem sido usado para medir a rigidez de hidrogel em trabalhos anteriores3,9,10. Primeiros trabalhos que demonstrou a importância da rigidez de substrato para o movimento de células utilizaram este método para determinar o hidrogel substrato rigidez3. Mais recentemente, microscopia confocal também tem sido usada para uma caracterização elegante10.
Aqui, apresentamos um protocolo passo a passo para preparar um substrato de silicone suave, acoplamento grânulos fluorescentes (e uma proteína da matriz extracelular como colágeno eu) só para a superfície superior, uma esfera de recuo e o topo usando superfície de imagem de fase e fluorescência de imagem, respectivamente e, finalmente, analisando as imagens para calcular o módulo de Young do substrato do silicone. O substrato de silicone suave preparado dessa maneira pode ser facilmente usado para experimentos de microscopia de força de tração. O uso de silicone duro (em vez de uma placa de Petri) como a base para o silicone macio também permite estudos de mechanobiology usando um trecho externo. Que tal se justifique, as considerações práticas necessárias para evitar possíveis complicações também são indicadas.

<table border="0" cellpadding="0" cellspacing="0" width="949">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:353px;">CY 52-276 A/B silicone elastomer kit</td>
			<td style="width:191px;">&#160;Dow Corning</td>
			<td style="width:144px;">CY 52-276</td>
			<td style="width:263px;">Store at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Scientific Pierce EDC</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td>PI22980</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Scientific&#160;Pierce Sulfo-NHS crosslinker</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td>PI-24510</td>
			<td>Store at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carboxyl fluorescent pink particles, 0.4-0.6 &#181;m, 2 mL</td>
			<td>Spherotech, Inc.</td>
			<td>CFP-0558-2</td>
			<td>Store at 4&#176;C, do not freeze</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.0 mm Acid washed Zirconium beads</td>
			<td style="width:191px;">OPS Diagnostics LLC</td>
			<td>BAWZ 1000-250-33</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:353px;">Deep UV chamber with ozone evacuator</td>
			<td style="width:191px;">Novascan Technologies, Inc.</td>
			<td style="width:144px;">PSD-UV4, OES-1000D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Wide field fluorescence microscope</td>
			<td style="width:191px;">Leica Microsystems</td>
			<td style="width:144px;">DMi8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Collagen I, from rat tail</td>
			<td style="width:191px;">Corning</td>
			<td style="width:144px;">354236</td>
			<td>Stock concentration = 4 mg/ml; store at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">ImageJ-NIH</td>
			<td style="width:191px;">N/A</td>
			<td style="width:144px;">N/A</td>
			<td>public-domain software</td>
		</tr>
	</tbody>
</table>

stiffness measurement of soft silicone substrates for mechanobiology studies using a widefield fluorescence microscope

Tecidos moles do corpo humano, normalmente, tem rigidez na faixa quilopascal (kPa). Nesse sentido, substratos flexíveis de silicone e hidrogel tem provados para ser útil substratos para cultivo de células em um microambiente física que imita parcialmente na vivo condições. Aqui, apresentamos um protocolo simples para caracterizar o módulo de Young de substratos de elásticos lineares isotrópicos normalmente utilizados para estudos de mechanobiology. O protocolo consiste em preparar um substrato de silicone macio em uma placa de Petri ou silicone duro, revestindo a superfície superior do substrato do silicone com grânulos fluorescentes, usando uma esfera de milímetro-escala para recuar a superfície superior (por gravidade), imagem latente da fluorescente grânulos na superfície do silicone recuado usando um microscópio de fluorescência e analisando as imagens resultantes para calcular o módulo de Young do substrato do silicone. A superfície superior do substrato de acoplamento com uma proteína de matriz extracelular moduli (além do que os grânulos fluorescentes) permite que o substrato do silicone a ser facilmente usado para chapeamento da célula e estudos posteriores usando experimentos de microscopia de força de tração. O uso de silicone duro, em vez de uma placa de Petri, como a base do silicone macio, permite o uso de mechanobiology estudos envolvendo trecho externo. Uma vantagem específica do presente protocolo é um microscópio de fluorescência widefield, que é comumente disponível em muitos laboratórios, os principais equipamentos necessários para este procedimento. Vamos demonstrar este protocolo medindo-se o módulo de Young de substratos de silicone suave de diferentes módulos elásticos.

Usando o protocolo detalhado acima, temos preparado silicone macio em uma placa de Petri de 35mm, curou-a 70 ° C por 30 min e acoplado microbeads fluorescente (e colágeno eu) para a superfície superior como esquematicamente representada na Figura 1. UV profundo tenha sido usado anteriormente para a proteína eventual acoplamento para substratos13. Note que (I) as condições de cura usadas aqui são específicas para este silic...

Watch this Scientific Journal Video about Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope at JoVE.com

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Substratos com rigidez na faixa de superfície são úteis para estudar a resposta das células à rigidez de microambiente fisiologicamente relevante. Usando apenas um microscópio de fluorescência widefield, o módulo de Young de gel de silicone suave pode ser determinado usando um recuo com uma esfera apropriada.

Medição da rigidez de substratos de Silicone macio para estudos de Mechanobiology, usando um microscópio de fluorescência Widefield

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

This method can be used to categorize substrates used to study key phenomena in mechanobiology, the cell migration and first generation. The main advantage of this technique is that it requires the use of a wide-field microscope, a piece of equipment that is commonly available in many labs. This method can be applied to measure the Young's modulus of silicon or polyacrylamide gel, as well as any of the soft isotropic linear elastic material.The substrate's prepared and characterized by this method can also be used for self-stretching experiments with the stiff silicone base. To fabricate the soft silicone substrate after weighting out about 1.75 grams each of components A and B from the soft silicone elastomer kit, add most of the weighed portion of component A to the B component polystyrene weighing tray and use an appropriate applicator stick to mix the two components together for five minutes. Transfer the mixture to a 35 millimeter Petri dish and allow the substrate to spread evenly across the Petri dish for a few minutes.Next, place the Petri dish in a vacuum chamber for 15 minutes to remove any air bubbles and pre-heat a hot plate. When the hot plate reaches 70 degrees Celsius, place a glass slide onto the hot plate, and place the Petri dish onto the glass side. Then, let the silicone cure at 70 degrees Celsius for 30 minutes.To couple the soft silicone with fluorescent beads, place the cured silicone in a deep UV chamber, five to ten centimeters away from the UV lamp and expose the sample to deep UV light for five minutes. While the silicone is being treated, dissolve 19 milligrams of EDC in 500 microliters of deionized water in a 1.5 milliliter microcentrifuge tube. And vortex the tube to dissolve the chemical.Next, add 500 microliters of deionized water to 11 milligrams of sulfo-NHS in a second 1.5 milliliter tube. And vortex to dissolve the chemical. Then, combine the EDC and sulfo-NHS solutions into a single microcentrifuge tube.Now, add 30 microliters of an appropriate suspension of fluoro-4 conjugated carboxylate-modified micro-beads and 0.02 milligrams of rat tail Collagen 1 to the mixture. After briefly vortexing, place a piece of Parafilm onto the lid of a second smaller diameter lid and transfer one milliliter of the EDC-NHS bead Collagen 1 mixture to the Parafilm. Invert the Petri dish containing the soft silicone over the mixture, supporting the dish on both sides with two glass slides, so that the soft silicone surface contacts the bead solution without directly touching the surface of the smaller diameter lid below.Then, cover the sample with aluminum foil for 30 minutes at room temperature, before washing the silicone two times, with two milliliters of PBS per wash and let the silicon cure upright in two milliliters of fresh PBS at 37 degrees Celsius overnight. The next morning, use pointed tweezers to drap five millimeter zirconium sphere indentors onto the soft silicone under at least one milliliter of PBS away from the edges of the silicone layer and at least five indentor diameters away from the location of the other indentors. Next, place the dish of soft silicon onto a wide-field microscope stage and use the 10x objective to obtain a phase image of a whole indentor without any visible defects.After saving the image, select the appropriate fluorescent channel for visualizing the fluorescent micro-beads. With the indentor center near the right edge of the frame, decrease the z-coordinates until the fluorescent micro-beads under the sphere indentor's center go just out of focus. Then, acquire a z-stack with an image for every 0.5 micrometer z-increment until the micro-beads in the top layer of the silicone near the left edge of the imaging frame come in and then out of focus.To calculate the silicone stiffness, first, open the indentor phase image in ImageJ and click analyze and set scale and check unit of length to confirm that the length is set to pixels. Using the line tool, click and hold onto a point on the indentor edge, moving the cursor to a point on the diametrically opposite edge, to measure the diameter of the indentor and note the length in pixels on the status bar of the ImageJ main window before releasing the cursor. Next, open the file menu and select import an image sequence to allow an image in the z-stack of fluorescent micro-bead images to be selected and click okay to open the stack.Using the line tool, draw a line across a well-defined micro-bead in one image and click analyze plot profile in live to obtain the updated line scan intensity across the bead, while selecting different frames. The frame that gives the highest value of the maximum intensity can then be selected as the frame in focus. Fluorescence images of the micro-beads in the top surface of the silicone taken at an x-y frame position, places the region under the indentor to the far-right side of the frame.The region on the far-left side of the frame is then selected as the region away from the indentor. In this representative set of intensity line scans measured across a single micro-bead, the focus varies in z-increments of 0.5 micrometers. The z-value corresponding to the in-focus image can then be objectively selected based on the z-value corresponding to the line scan with the highest maximum intensity.Here, the Young's modulus of the silicone for various A:B component mixture ratios, was obtained based on the method as just demonstrated. After watching this video, you should have a good understanding of how to prepare soft silicone substrates, cup the fluorescent beads to them, and measure the stiffness of the substrate using a wide-field fluorescent microscope.

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