All authors gratefully acknowledge funding support from the National Research Foundation of Singapore through the Singapore-MIT Alliance for Research and Technology (SMART) BioSystems and Micromechanics (BioSyM) interdisciplinary research group. Authors Dr. Jagielska and Dr. Van Vliet also gratefully acknowledge funding and support from the Saks-Kavanaugh Foundation. The authors thank William Ong and Dr. Sing Yian Chew from Nanyang Technological University, Singapore, for providing rat oligodendrocyte progenitor cells for some experiments described in this work, and the authors thank Dr. G. V. Shivashankar from Mechanobiology Institute, National University of Singapore, Singapore, for discussions about nuclear area fluctuations.

1. Design of the single-well polydimethylsiloxane mold for high-resolution imaging
NOTE: The mold for manufacturing polydimethylsiloxane plates is designed with the following features to enable imaging with a 100x oil immersion lens and a correct fit in the custom-build strain device (Figure 1A,B).
<ol>
	<li>Keep the overall plate dimensions such that it fits in the clamps of the strain device. 
	NOTE: Here, they are 60 mm x 73 mm.</li>
	<li>Make a cell compartment or well that is significantly smaller than the whole plate&#8217;s lateral dimensions, to avoid arcing (out-of-plane bending) effects that happen at the unclamped edges of the polydimethylsiloxane plate during applied strain. 
	NOTE: Here, the compartment was designed as a 23 mm x 23 mm square.</li>
	<li>Keep the depth of the cell compartment as minimal as possible (not more than 80 &#181;m), to enable focusing with the 100x oil immersion lens through the cover glass placed on top of the compartment, but make it sufficient enough to contain media and to avoid contacting or squeezing the cells.</li>
	<li>Raise the cell compartment on a square with a height of 3 mm, to bring the cells closer to the lens in the inverted microscope setup to enable easy focusing. 
	NOTE: Molds with the above features can be manufactured by many techniques including, for example, milled aluminum. Alternatively, master molds can also be assembled by using acrylic sheets cut with a laser cutter, cover glass #0 for the cell compartment, and super glue. This master mold is used to make a polydimethylsiloxane imprint, which is then used to make working molds using a casting resin.</li>
</ol>
2. Fabrication of single-well polydimethylsiloxane plates and square compartments
<ol>
	<li>Mix polydimethylsiloxane base and curing agent in a ratio of 20:1 (by weight) in a disposable cup. Weigh a total of 20 g of polydimethylsiloxane per mold (for making one polydimethylsiloxane plate) and 150 g of polydimethylsiloxane per 150 mm-in-diameter plastic dish (for making one batch of square compartments).</li>
	<li>Leave the polydimethylsiloxane mix in a vacuum degasser at -0.8 bar for 30 min (or until all bubbles are removed).</li>
	<li>Pour the polydimethylsiloxane mix into the molds (for plates) or into 150 mm plastic dishes (for square compartments).</li>
	<li>Remove any additional bubbles at this stage, either by blowing air (by mouth) or degassing the polydimethylsiloxane-filled molds/dishes again at -0.8 bar vacuum for 30 min.</li>
	<li>Leave the molds/dishes in an 80 &#176;C oven on a leveling table for&#160;2 h.</li>
	<li>Carefully remove the molds/dishes from the oven and let them cool down to room temperature. Gently peel out the cured polydimethylsiloxane after carefully cutting the edges with a blade (Figure 1C).</li>
	<li>On the 150 mm-diameter polydimethylsiloxane, draw a 2 cm x 2 cm grid with a marker. Within each square, draw a 1 cm x 1 cm square, leaving a margin of 0.5 cm on all sides. Using a blade, carefully cut along the lines to obtain square compartments (Figure 2A).</li>
	<li>Clean the polydimethylsiloxane plates/square compartments by incubating them in 100% acetone for 4&#160;h, followed by 100% ethanol for 4&#160;h, followed by autoclaved water for 4&#160;h.</li>
	<li>Place the plates/square compartments on paraffin film backing paper (not the paraffin film but the paper) inside a 150 mm diameter plastic dish (Figure 2C).</li>
	<li>Leave the polydimethylsiloxane plate in the 80 &#176;C oven to dry for 4&#160;h.</li>
	<li>Seal the 150 mm-diameter dishes containing polydimethylsiloxane plates and container squares with paraffin film, and store them in a cold room until further use.</li>
</ol>
3. Functionalization of polydimethylsiloxane plates
<ol>
	<li>Remove the paraffin film, remove the cover of the plastic dish, and place the polydimethylsiloxane plates and square compartments under ultraviolet light for 30 min.</li>
	<li>Plasma-treat (at 150 W for 5 min) the polydimethylsiloxane plates (with the cell compartment facing up) without the square compartments, to make the culture surface hydrophilic.</li>
	<li>Immediately place the square compartments onto the plasma-treated surface (Figure 2B) and press manually to temporarily stick the two together. 
	NOTE: Do not plasma-treat the square compartments, or else they will stick strongly to the polydimethylsiloxane plate and will not come off easily when they must be peeled off during imaging.</li>
	<li>Add 200 &#181;L of 100 mM (3-aminopropyl)triethoxysilane to the well for 2 h to introduce &#8211;NH2 groups to the polydimethylsiloxane surface. To make a 100 mM solution, dissolve 234 &#181;L of pure (3-aminopropyl)triethoxysilane in 10 mL of water. After a 2 h incubation, wash the wells 3x with deionized water.</li>
	<li>Weigh 2 mg of the molecular crosslinker bissulfosuccinimidyl suberate and dissolve it in 700 &#181;L of 1 M (4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid buffer (pH 8.0) and 2.8 mL of water. 
	NOTE: This will give a 1 mM bissulfosuccinimidyl suberate solution in 200 mM (4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid buffer. Note that a 50 mM concentration buffer also works well.
	<ol>
		<li>Add 135 &#181;L of this solution and 15 &#181;L of 1 mg/mL fibronectin to the well in each polydimethylsiloxane plate for 4 h at room temperature.</li>
	</ol>
	</li>
	<li>Wash 3x with phosphate-buffered saline solution. Add 500 &#181;L of phosphate-buffered saline solution to the well. Cover the 150 mm-diameter dish containing the polydimethylsiloxane with paraffin film and store it in a cold room, until further use.</li>
</ol>
4. Functionalization of plastic dishes and flasks
<ol>
	<li>Incubate plastic dishes and flasks with a 5 &#181;g/mL solution of poly-D-lysine (PDL, in sterile water) for 1 h. Add 1.5 mL of this solution (ligand) per 35 mm-diameter dish, 3 mL of it per 60 mm-diameter dish, and 10 mL per 75 cm2 culture flask.</li>
	<li>Wash the dishes and flasks 2x with sterile water.</li>
	<li>Leave them to dry in the biosafety cabinet for 1 h (or until dry) and store them in the cold room at 4 &#176;C until further use.</li>
</ol>
5. Proliferation and differentiation medium for murine oligodendrocyte progenitor cells
<ol>
	<li>Prepare 50 mL of 100x solutions of the proliferation and differentiation medium, make aliquots of 2.5 mL, and store them at -80 &#176;C. 
	NOTE: The composition of proliferation medium is 0.1 mg/mL bovine serum albumin, 62 ng/mL progesterone, 16 &#181;g/mL putrescine, 5 &#181;g/mL insulin, 50 &#181;g/mL holo-transferrin, 5 ng/mL sodium selenite, 1x penicillin-streptomycin, 10 ng/mL platelet-derived growth factor, and 10 ng/mL fibroblast growth factor in Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) with high glucose and pyruvate. While bovine serum albumin, progesterone, putrescine, and sodium selenite can be constituted and stored at 100x, insulin, holo-transferrin, and penicillin-streptomycin must be added fresh while preparing the 1x solution. The growth factors must be constituted at 10 &#181;g/mL and must be added fresh to the cells every day (1 &#181;L/mL of medium). The composition of the differentiation medium is 0.1 mg/mL bovine serum albumin, 62 ng/mL progesterone, 16 &#181;g/mL putrescine, 5 &#181;g/mL insulin, 50 &#181;g/mL holo-transferrin, 5 ng/mL sodium selenite, 1x penicillin-streptomycin, 400 ng/mL triiodothyronine, 400 ng/mL L-thyroxine, and 0.5% fetal bovine serum in DMEM with high glucose and pyruvate. Triiodothyronine and L-thyroxine can be constituted with other reagents and stored at 100x. Insulin, holo-transferrin, and penicillin-streptomycin must be added fresh while preparing the 1x solution.</li>
	<li>For preparing 50 mL of 100x proliferation medium, dissolve 510 mg of bovine serum albumin in 17 mL of Dulbecco&#8217;s medium, 310 &#181;g of progesterone in 31 &#181;L of ethanol, 80 mg of putrescine in 500 &#181;L of Dulbecco&#8217;s medium, 25 &#181;g of sodium selenite in 10 &#181;L of Dulbecco&#8217;s medium, and full DMEM up to 50 mL</li>
	<li>For preparing 50 mL of 100x solution of differentiation medium, additionally dissolve 2 mg of triiodothyronine in 40 &#181;L of sodium hydroxide and 2 mg of L-thyroxine in 40 &#181;L of sodium hydroxide, then fill up the solution to 50 mL with Dulbecco&#8217;s medium. Filter the solution through a sterile disposable filter unit, aliquot, and store the aliquots at -80 &#176;C.</li>
	<li>For preparing 250 mL of 1x proliferation/differentiation medium, mix 2.5 mL of 100x solution of proliferation/differentiation medium with 12.5 mg of holo-transferrin, 125 &#181;L of 10 mg/mL insulin, and 2.5 mL of 100x penicillin-streptomycin. Fill up the solution to 250 mL with Dulbecco&#8217;s medium and filter it through a sterile disposable filter unit. 
	NOTE: The growth factors must be added fresh and directly to the cell culture, not to the whole batch of reconstituted medium.</li>
</ol>
6. Cell culture
<ol>
	<li>Start with oligodendrocyte progenitor cells suspended in proliferation medium. Seed these cells at a density of 35,000 cells/cm2 onto PDL-coated plastic surfaces (dishes or flasks, see section 4). Adjust the volume of the proliferation medium to 3 mL per 10 cm2 of surface area of plastic substratum; a lower volume of medium may lead to cell clumping arising from surface tension forces, while a higher volume of medium in the dishes may cause spillage.</li>
	<li>Add growth factors (platelet-derived growth factor and fibronectin growth factor) every day, maintaining their concentration at 10 ng/mL and change half of the proliferation medium on alternate days.</li>
	<li>On the third day, detach the cells from the plastic surface using a gentle cell detachment solution (e.g., accutase): remove the medium, wash 1x with phosphate-buffered saline solution, add 1 mL of detachment solution per 20 cm2 area, leave the dish/flask in an incubator at 37 &#176;C for 10 min, and gently tap it until all cells have detached (look under a microscope).</li>
	<li>Pipette using a 200 &#181;L tip to break clumps into single cells, transfer them to a 50 mL tube, dilute them in proliferation medium, spin at 0.2 x g for 10 min, discard the supernatant, and resuspend the pellet in proliferation medium to make a total volume of 1 mL. Count the cells using a cytometer.</li>
	<li>Wash the fibronectin-functionalized polydimethylsiloxane plates 2x, 15 min per wash, with proliferation medium.</li>
	<li>Seed 35,000 cells per polydimethylsiloxane plate in 700 &#181;L of proliferation medium.</li>
	<li>Add a plasmid construct of H2B-GFP for labeling histone H2B with green fluorescent protein. 
	NOTE: Here, 1.4 &#181;L of CellLight H2B-GFP BacMam2 transfection mix was added to each plate (2 &#181;L per 50,000 cells).</li>
	<li>After 24 h, mount the polydimethylsiloxane samples with cells to be stretched on the uniaxial strain device (as shown in Figure 2D). Change the medium in all samples to differentiation medium.</li>
	<li>To strain the samples, measure the unstrained cell compartment length (Figure 3A) and turn the stage micrometer screw to increase the cell compartment length by the desired amount (e.g., 10%, see Figure 3B). Leave the stretched and unstretched polydimethylsiloxane samples in the incubator at 37 &#176;C until imaging.</li>
</ol>
7. Imaging
<ol>
	<li>Turn on the microscope. Set the microscope incubator temperature to 37 &#176;C.</li>
	<li>Bring the objective to be used (100x oil immersion) to the central position as the objective turret will not be accessible later. Unscrew the objective and screw it back together with an objective ring (Figure 4A) so that the objective can be brought closer to the cells. If an oil objective is being used here, add a drop of oil at this step.</li>
	<li>Synthesize beforehand (via 3D printing or machining) a plastic or metal holder that has two important features&#8212;an angled window and a step (Figure 4B). 
	NOTE: The window supports a thickness #0 glass coverslip that will hold the medium while the strain device is in an inverted state. The angled cut at the window edges (Figure 4D) allows the objective to come closer to the glass coverslip. The step in the holder allows us to bring the stretched polydimethylsiloxane further down, closer to the objective.</li>
	<li>Place a glass coverslip (thickness #0, with dimensions of 25 mm x 25 mm or 35 mm in diameter) onto the top surface of the holder by spreading vacuum grease at the periphery of the window (Figure 4C), and tape the plastic window onto the microscope stage (Figure 4D and Figure 5A).</li>
	<li>Screw a z-translation stage onto the microscope stage and move it to the topmost z-position (Figure 5A).</li>
	<li>Remove 500 &#181;L of the medium from the stretched polydimethylsiloxane plate to be imaged and add this medium onto the glass coverslip in the white plastic window.</li>
	<li>Carefully detach the square compartment from the polydimethylsiloxane plate with sterile tweezers.</li>
	<li>Hold the strain device in an upright position (cells facing up) above the white plastic window and carefully invert the strain device so as to let any extra medium drop directly in the middle of the glass coverslip (Figure 5B) (cells facing down).</li>
	<li>Place the bottom part of the strain device onto the z-translation stage with the double-sided tape (Figure 5C,D).</li>
	<li>While looking through the eyepiece under brightfield, slowly bring the strain device down (Figure 5E) (by lowering the z-translation stage) and move the objective up to focus on the cells.
	<ol>
		<li>Perform this step extremely slowly, step by step. If the strain device presses too much onto the coverslip, the cells can get compressed (causing them to die) and if the objective presses too much onto the coverslip, the coverslip can break (causing medium to leak and spill onto the objective).</li>
	</ol>
	</li>
	<li>Scan the polydimethylsiloxane plate in x- and y-directions to find a cell that has a fluorescent nucleus (under epifluorescence with 488 nm of wavelength excitation) and appropriate cell morphology (under brightfield).</li>
	<li>If the lateral displacement of the microscope stage does not affect the vertical focusing too much, select multiple regions of interests using a multipoint feature on the software. Sometimes, the focusing is very sensitive to any lateral displacement of the stage. In such cases, image one cell at a time. Mark the x-, y-, and z-positions for each cell of interest.</li>
	<li>Record wide-field (or open-pinhole) images of the nucleus with 488 nm of wavelength excitation and of the cell with brightfield excitation at intervals of 30 s per frame for a total duration of at least 30 min.</li>
</ol>
8. Data analysis
<ol>
	<li>Nuclear fluctuations
	<ol>
		<li>Open the sequence of nucleus images (Figure 6A) in an image analysis software and threshold the nucleus time-lapse images (for example, use the Threshold command in ImageJ or the Graythresh command in the software MATLAB).</li>
		<li>Get the nucleus area (in pixels) as a function of time, plot it in a data plotting software (Figure 6B), and fit a third order polynomial to the data (Figure 6C) (for example, use the Analysis | Fitting | Polynomial Fit command in Origin or the Polyfit command in the software MATLAB).</li>
		<li>Subtract the value of the fitted polynomial from the actual area (in pixels) for each time point. This corrected area is known as the residual area. 
		NOTE: This process removes any increasing or decreasing trend in the data coming from instrumental errors and is called detrending.
		<ol>
			<li>Calculate the percentage of residual area by dividing the residual area at each time point with the value of the fitted polynomial at that time point (Figure 6D).</li>
		</ol>
		</li>
		<li>Calculate the standard deviation of the residual area time series. This standard deviation denotes the amplitude of nuclear fluctuations.</li>
	</ol>
	</li>
	<li>Data plotting and statistical analysis
	<ol>
		<li>Calculate the amplitude of nuclear fluctuations as a mean of at least 20 nuclei per condition.</li>
		<li>Conduct a one-way analysis-of-variance statistical test with Bonferroni correction to determine whether there is a significant difference in the amplitude of fluctuations as a function of conditions of interest (for example, with and without applied strain).</li>
	</ol>
	</li>
</ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+plasticity+of+the+nucleus+in+stem+cell+differentiation.">Physical plasticity of the nucleus in stem cell differentiation.</a> Proceedings of the National Academy of Sciences of the United States of America. 1, 0 (2007).</li><li>Talwar, S., Kumar, A., Rao, M., Menon, G. I., Shivashankar, G. 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The authors have nothing to disclose.

A device has previously1 been designed for the application of extracellular tensile strain on adherent cells. The design of the polydimethylsiloxane substratum in that work was sufficient for biochemical assays, as well as the low-resolution imaging of stretched cells. In this work, the substratum was redesigned, and a novel imaging configuration that facilitates high-resolution subcellular live cell imaging was introduced. The advantages of this system are numerous: it can be built in-lab using s...

Cells and tissues in the body are subjected to various mechanical cues, including tensile strains. However, the effects of these cues on the biology of neural cells have not yet been studied extensively and understood fully. In the central nervous system, sources of mechanical strain include developmental growth1,2,3,4, physiological processes such as spinal cord bending, blood and cerebrospinal fluid pulsation, and pathological conditions such as trauma, axon swelling, glial scarring, or tumor growth5,6,7,8. It is worth investigating how tensile strain affects the differentiation of oligodendrocytes and the subsequent myelination of axons, which is a critical process in the vertebrate central nervous system. Using a custom-designed strain device and elastomeric multiwell plates, previous works9,10 have shown that static uniaxial strain can increase oligodendrocyte differentiation via global changes in gene expression10. To gain further understanding of the mechanisms of strain mechanotransduction in these cells, the previous experimental apparatus must be redesigned as described here, to enable high-resolution fluorescence imaging of nuclear dynamics in living cells under strain. Specifically, a single-well polydimethylsiloxane plate is developed, and the imaging configuration is redesigned to allow for the time-lapse imaging of live cells under strain using a 100x oil immersion lens. To eliminate the negative optical effects of polydimethylsiloxane in the light pathway, cells are imaged not through the polydimethylsiloxane plate, but in the inverted position, through the cover glass covering the cell compartment. Using this new imaging design, hundreds of high-resolution time-lapse movies are recorded, of individual cell nuclei within intact adherent cells, where chromatin is labeled by tagging histone H2B to green fluorescent protein. These movies demonstrate that tensile strain induces changes in chromatin structure and dynamics that are consistent with the progression of oligodendrocyte differentiation.
Live cell imaging under applied strain is technically challenging and requires a device design that is compatible with the microscope system. The custom design described here presents an inexpensive alternative to commercial solutions. Its dimensions enable its installation on microscope stages and live cell imaging at high spatial resolution during applied strain. The imaging setup is designed to facilitate live cell imaging using a 100x oil immersion lens with the highest clarity, through the cover glass, not through the layer of polydimethylsiloxane plate which otherwise decreases the image quality and is common in most imaging setups under strain. The device, with a mounted plate containing cells, can also be stored easily in the incubator. This device is designed to apply uniaxial strain to substrata that facilitate adherent cell culture and maintain a stable and uniform strain over multiple days. The setup described here can be used for the high-resolution imaging of various adherent cell types under strain, making it applicable to mechanotransduction studies in many fields of cell mechanobiology.

<table><tbody><tr><td>&lt;strong&gt;Primary cells&lt;/strong&gt;</td><td></td><td></td><td>Primary Oligodendrocyte Progenitor Cells isolated from Neonatal Rats&lt;br/&gt; were provided by Dr. S. Y. Chew&#039;s lab at Nanyang Technological University</td></tr><tr><td>&lt;strong&gt;Media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM high&amp;nbsp; glucose</td><td>Gibco</td><td>11996-065-500ml</td><td></td></tr><tr><td>Pen/Strep</td><td>Gibco</td><td>15070-063-100ml</td><td></td></tr><tr><td>FGF</td><td>Prospec</td><td>CYT-608-50ug</td><td></td></tr><tr><td>PDGF</td><td>Prospec</td><td>CYT-776-10ug</td><td></td></tr><tr><td>&lt;strong&gt;Media Stock components&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A9418-10G</td><td></td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P8783-1G</td><td></td></tr><tr><td>Putrescine</td><td>Sigma</td><td>P5780-5G</td><td></td></tr><tr><td>Sodium Selenite</td><td>Sigma</td><td>S5261-10G</td><td></td></tr><tr><td>Tri-iodothyronine</td><td>Sigma</td><td>T6397-100MG</td><td></td></tr><tr><td>Thyroxine</td><td>Sigma</td><td>T1775-100MG</td><td></td></tr><tr><td>Holo-Transferrin</td><td>Sigma</td><td>T0665-500MG</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>I9278</td><td></td></tr><tr><td>Mold and PDMS plate</td><td></td><td></td><td></td></tr><tr><td>PDMS - Dow Corning Sylgard 184</td><td>Ellsworth</td><td>184 SIL ELAST KIT 0.5KG</td><td></td></tr><tr><td>Mold - Liquid Plastic</td><td>Smooth-On</td><td>Smooth Cast 310 - Trail Size</td><td></td></tr><tr><td>Substrate Coatings</td><td></td><td></td><td></td></tr><tr><td>poly-D-lysine</td><td>Sigma</td><td>P6407-5MG</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma</td><td>F1141-2MG</td><td></td></tr><tr><td>&lt;strong&gt;Histone staining&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CellLigt H2B-GFP BacMam</td><td>Molecular Probes</td><td>C10594</td><td></td></tr><tr><td>&lt;strong&gt;Surface functionalization&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>APTES</td><td>Sigma</td><td>A3648-100ml</td><td></td></tr><tr><td>BS3</td><td>Covachem</td><td>13306-100mg</td><td></td></tr><tr><td>HEPES buffer pH 8.0</td><td>Alfa Aesar</td><td>J63578-AK-250ml</td><td></td></tr><tr><td>&lt;strong&gt;Cell detachment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Accutase</td><td>Invitrogen</td><td>A1110501-100ml</td><td></td></tr></tbody></table>

high-resolution imaging of nuclear dynamics in live cells under uniaxial tensile strain

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the effect of applied extracellular tensile strain on cell populations in vitro via biochemical assays, a device has previously been designed which can be fabricated simply and is small enough to fit inside tissue culture incubators, as well as on top of microscope stages. However, the previous design of the polydimethylsiloxane substratum did not allow high-resolution subcellular imaging via oil-immersion objectives. This work describes a redesigned geometry of the polydimethylsiloxane substratum and a customized imaging setup that together can facilitate high-resolution subcellular imaging of live cells while under applied strain. This substratum can be used with the same, earlier designed device and, hence, has the same advantages as listed above, in addition to allowing high-resolution optical imaging. The design of the polydimethylsiloxane substratum can be improved by incorporating a grid which will facilitate tracking the same cell before and after the application of strain. Representative results demonstrate high-resolution time-lapse imaging of fluorescently labeled nuclei within strained cells captured using the method described here. These nuclear dynamics data give insights into the mechanism by which applied tensile strain promotes differentiation of oligodendrocyte progenitor cells.&#160;

Recent work aimed at investigating the effect of tensile strain on oligodendrocytes10 showed that a 10% uniaxial tensile strain promotes the differentiation of oligodendrocyte progenitor cells by global changes in gene expression. The mechanism behind these changes in gene expression can be probed via the imaging of subcellular parameters, such as the cytoskeleton structure, transcription factor localization, nuclear dynamics, and chromatin organization. However, t...

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High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain

Using a previously designed device to apply mechanical strain to adherent cells, this paper describes a redesigned substratum geometry and a customized apparatus for high-resolution single-cell imaging of strained cells with a 100x oil immersion objective.

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the ...

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Research

JoVE Journal

Bioengineering

7.8K Views.  Singapore-MIT Alliance for Research and Technology, CREATE. We hebben eerder een apparaat ontworpen om mechanische belasting toe te passen op aanhangende cellen. In het huidige werk hebben we de geometrie van het substratum opnieuw ontworpen en de setup aangepast voor hoge resolutie beeldvorming van deze stamcellen. Het belangrijkste voordeel van dit cel-stretching systeem is dat het een hoge resolutie subcellulaire beeldvorming biedt bij 100x van de stamcellen.Het aantonen van de substratum fabricage zal Vicki zijn, een onderzoeksassistent van...

Video: High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain

A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton

The mechanical properties of the nucleus determine its response to mechanical forces generated in cells. Because the nucleus is molecularly continuous with the cytoskeleton, methods are needed to probe its mechanical behavior in adherent cells. Here, we discuss the direct force probe (DFP) as a tool to apply force directly to the nucleus in a living adherent cell. We attach a narrow micropipette to the nuclear surface with suction. The micropipette is translated away from the nucleus, which causes the nucleus to deform and translate. When the restoring force is equal to the suction force, the nucleus detaches and elastically relaxes. Because the suction pressure is precisely known, the force on the nuclear surface is known. This method has revealed that nano-scale forces are sufficient to deform and translate the nucleus in adherent cells, and identified cytoskeletal elements that enable the nucleus to resist forces. The DFP can be used to dissect the contributions of cellular and nuclear components to nuclear mechanical properties in living cells.

In this protocol, we describe a micropipette method to directly apply a controlled force to the nucleus in a living cell. This assay allows interrogation of nuclear mechanical properties in the living, adherent cell.

The mechanical properties of the nucleus determine its response to mechanical forces generated in cells. Because the nucleus is molecularly continuous ...

Biochemistry

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

Live Cell Imaging during Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin rods are a hallmark of a conserved, inducible Actin Stress Response (ASR) that accompanies human pathologies, including neurodegenerative disease. Previously, we showed that the ASR contributes to morphogenesis failures and reduced viability of developing embryos. This protocol allows the continued study of mechanisms underlying actin rod assembly and the ASR in a model system that is highly amenable to imaging, genetics and biochemistry. Embryos are collected and mounted on a coverslip to prepare them for injection. Rhodamine-conjugated globular actin (G-actinRed) is diluted and loaded into a microneedle. A single injection is made into the center of each embryo. After injection, embryos are incubated at elevated temperature and intranuclear actin rods are then visualized by confocal microscopy. Fluorescence recovery after photobleaching (FRAP) experiments may be performed on the actin rods; and other actin-rich structures in the cytoplasm can also be imaged. We find that G-actinRed polymerizes like endogenous G-actin and does not, on its own, interfere with normal embryo development. One limitation of this protocol is that care must be taken during injection to avoid serious injury to the embryo. However, with practice, injecting G-actinRed into Drosophila embryos is a fast and reliable way to visualize actin rods and can easily be used with flies of any genotype or with the introduction of other cellular stresses, including hypoxia and oxidative stress.

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The goal of this protocol is to inject Rhodamine-conjugated globular actin into Drosophila embryos and image intranuclear actin rod assembly following heat stress.

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin ...

Developmental Biology

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.

Here, we present a protocol to investigate the intracellular mechanical properties of isolated embryonic zebrafish cells in three-dimensional confinement with direct force measurement by an optical trap.

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Biology

DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical for regulating many processes ranging from development to immunology. Therefore, developing the tools to study these forces at the molecular scale is critical. Our group developed a suite of molecular tension sensors to quantify and visualize the forces generated by cells and transmitted to specific ligands. The most sensitive class of molecular tension sensors are comprised of nucleic acid stem-loop hairpins. These sensors use fluorophore-quencher pairs to report on the mechanical extension and unfolding of DNA hairpins under force. One challenge with DNA hairpin tension sensors is that they are reversible with rapid hairpin refolding upon termination of the tension and thus transient forces are difficult to record. In this article, we describe the protocols for preparing DNA tension sensors that can be "locked" and prevented from refolding to enable "storing" of mechanical information. This allows for the recording of highly transient piconewton forces, which can be subsequently "erased" by the addition of complementary nucleic acids that remove the lock. This ability to toggle between real-time tension mapping and mechanical information storing reveals weak, short-lived, and less abundant forces, that are commonly employed by T cells as part of their immune functions.

This paper describes a detailed protocol for using DNA-based tension probes to image the receptor forces applied by immune cells. This approach can map receptor forces &gt;4.7pN in real-time and can integrate forces over time.

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical ...

Injection of Porcine Adipose Tissue-Derived Stroma Cells via Waterjet Technology

Urinary incontinence (UI) is a highly prevalent condition characterized by the deficiency of the urethral sphincter muscle. Regenerative medicine branches, particularly cell therapy, are novel approaches to improve and restore the urethral sphincter function. Even though injection of active functional cells is routinely performed in clinical settings by needle and syringe, these approaches have significant disadvantages and limitations. In this context, needle-free waterjet (WJ) technology is a feasible and innovative method that can inject viable cells by visual guided cystoscopy in the urethral sphincter. In the present study, we used WJ to deliver porcine adipose tissue-derived stromal cells (pADSCs) into cadaveric urethral tissue and subsequently investigated the effect of WJ delivery on cell yield and viability. We also assessed the biomechanical features (i.e., elasticity) by atomic force microscopy (AFM) measurements. We showed that WJ delivered pADSCs were significantly reduced in their cellular elasticity. The viability was significantly lower compared to controls but is still above 80%.

We present a method of cell injection via needle free waterjet technology coupled with a sequela of post-delivery investigations in terms of cellular viability, proliferation, and elasticity measurements.

Urinary incontinence (UI) is a highly prevalent condition characterized by the deficiency of the urethral sphincter muscle. Regenerative medicine ...

Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear positioning occurs during skeletal muscle differentiation. Muscle fibers (myofibers) are multinucleated cells formed by the fusion of muscle precursor cells (myoblasts) derived from muscle stem cells (satellite cells) that undergo proliferation and differentiation. Correct nuclear positioning within myofibers is required for the proper muscle regeneration and function. The common procedure to assess myoblast differentiation and myofiber formation relies on fixed cells analyzed by immunofluorescence, which impedes the study of nuclear movement and cell behavior over time. Here, we describe a method for the analysis of myoblast differentiation and myofiber formation by live cell imaging. We provide a software for automated nuclear tracking to obtain a high-throughput quantitative characterization of nuclear dynamics and myoblast behavior (i.e., the trajectory) during differentiation and fusion.

Skeletal muscle differentiation is a highly dynamic process, which particularly relies on nuclear positioning. Here, we describe a method to track nuclei movements by live cell imaging during myoblast differentiation and myotube formation and to perform a quantitative characterization of nuclei dynamics by extracting information from automatic tracking.

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear ...

High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain

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