AFM analysis was performed on instrumentation supported by the Pennsylvania Muscle Institute and the Institute for Translational Medicine and Therapeutics, Perelman School of Medicine, the University of Pennsylvania. This work was supported by NIH grants HL62250 and AG047373. YHB was supported by post-doctoral fellowship from the American Heart Association.

<ol>
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F., 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=Arterial+Stiffness+and+Cardiovascular+Events:+The+Framingham+Heart+Study.">Arterial Stiffness and Cardiovascular Events: The Framingham Heart Study.</a> Circulation. 121, 505-511 (2010).</li><li>Sutton-Tyrrell, K., 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=Elevated+Aortic+Pulse+Wave+Velocity,+a+Marker+of+Arterial+Stiffness,+Predicts+Cardiovascular+Events+in+Well-Functioning+Older+Adults.">Elevated Aortic Pulse Wave Velocity, a Marker of Arterial Stiffness, Predicts Cardiovascular Events in Well-Functioning Older Adults.</a> Circulation. 111, 3384-3390 (2005).</li><li>Klein, E. A., 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=Cell-Cycle+Control+by+Physiological+Matrix+Elasticity+and+In+Vivo+Tissue+Stiffening.">Cell-Cycle Control by Physiological Matrix Elasticity and In Vivo Tissue Stiffening.</a> Curr Biol. 19, 1511-1518 (2009).</li><li>Bae, Y. 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=A+FAK-Cas-Rac-lamellipodin+signaling+module+transduces+extracellular+matrix+stiffness+into+mechanosensitive+cell+cycling.">A FAK-Cas-Rac-lamellipodin signaling module transduces extracellular matrix stiffness into mechanosensitive cell cycling.</a> Sci signal. 7, ra57 (2014).</li><li>Thyberg, J., Hedin, U., Sj&#246;lund, M., Palmberg, L., Bottger, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+differentiated+properties+and+proliferation+of+arterial+smooth+muscle+cells.">Regulation of differentiated properties and proliferation of arterial smooth muscle cells.</a> Arterioscler Thromb Vasc Biol. 10, 966-990 (1990).</li><li>Owens, G. K., Kumar, M. S., Wamhoff, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Regulation+of+Vascular+Smooth+Muscle+Cell+Differentiation+in+Development+and+Disease.">Molecular Regulation of Vascular Smooth Muscle Cell Differentiation in Development and Disease.</a> Physiol Rev. 84, 767-801 (2004).</li><li>Huynh, 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=Age-Related+Intimal+Stiffening+Enhances+Endothelial+Permeability+and+Leukocyte+Transmigration.">Age-Related Intimal Stiffening Enhances Endothelial Permeability and Leukocyte Transmigration.</a> Sci Transl Med. 3, 112ra122 (2011).</li><li>Safar, M. E., Levy, B. I., Struijker-Boudier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Perspectives+on+Arterial+Stiffness+and+Pulse+Pressure+in+Hypertension+and+Cardiovascular+Diseases.">Current Perspectives on Arterial Stiffness and Pulse Pressure in Hypertension and Cardiovascular Diseases.</a> Circulation. 107, 2864-2869 (2003).</li><li>Raffetto, J. D., Khalil, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinases+and+their+inhibitors+in+vascular+remodeling+and+vascular+disease.">Matrix metalloproteinases and their inhibitors in vascular remodeling and vascular disease.</a> Biochem Pharmacol. 75, 346-359 (2008).</li><li>Muller, D. J., Dufrene, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscopy+as+a+multifunctional+molecular+toolbox+in+nanobiotechnology.">Atomic force microscopy as a multifunctional molecular toolbox in nanobiotechnology.</a> Nat Nanotechnol. 3, 261-269 (2008).</li><li>Hsu, B. Y., Bae, Y. H., Mui, K. L., Liu, S. -. L., Assoian, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apolipoprotein+E3+Inhibits+Rho+to+Regulate+the+Mechanosensitive+Expression+of+Cox2.">Apolipoprotein E3 Inhibits Rho to Regulate the Mechanosensitive Expression of Cox2.</a> PLoS ONE. 10, e0128974 (2015).</li><li>Johnson, K. L., Kendall, K., Roberts, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+Energy+and+the+Contact+of+Elastic+Solids.">Surface Energy and the Contact of Elastic Solids.</a> Proc R Soc Lond A. 324, 301-313 (1971).</li><li>Derjaguin, B. V., Muller, V. M., Toporov, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+contact+deformations+on+the+adhesion+of+particles.">Effect of contact deformations on the adhesion of particles.</a> J Colloid Interface Sci. 53, 314-326 (1975).</li><li>Hutter, J. L., Bechhoefer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calibration+of+atomic-force+microscope+tips.">Calibration of atomic-force microscope tips.</a> Rev Sci Instrum. 64, 1868-1873 (1993).</li><li>Pries, A. R., Secomb, T. W., Gaehtgens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+surface+layer.">The endothelial surface layer.</a> Pflugers Arch EJP. 440, 653-666 (2000).</li><li>Pogoda, K., 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=Depth-sensing+analysis+of+cytoskeleton+organization+based+on+AFM+data.">Depth-sensing analysis of cytoskeleton organization based on AFM data.</a> Eur Biophys J. 41, 79-87 (2012).</li><li>Mendez, M. G., Restle, D., Janmey, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vimentin+Enhances+Cell+Elastic+Behavior+and+Protects+against+Compressive+Stress.">Vimentin Enhances Cell Elastic Behavior and Protects against Compressive Stress.</a> Biophys J. 107, 314-323 (2014).</li><li>Moreno-Flores, S., Benitez, R., Md Vivanco, ., Toca-Herrera, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+relaxation+and+creep+on+living+cells+with+the+atomic+force+microscope:+a+means+to+calculate+elastic+moduli+and+viscosities+of+cell+components.">Stress relaxation and creep on living cells with the atomic force microscope: a means to calculate elastic moduli and viscosities of cell components.</a> Nanotechnology. 21, 445101 (2010).</li><li>Darling, E. M., Topel, M., Zauscher, S., Vail, T. P., Guilak, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscoelastic+properties+of+human+mesenchymally-derived+stem+cells+and+primary+osteoblasts,+chondrocytes,+and+adipocytes.">Viscoelastic properties of human mesenchymally-derived stem cells and primary osteoblasts, chondrocytes, and adipocytes.</a> J Biomech. 41, 454-464 (2008).</li><li>Dimitriadis, E. K., Horkay, F., Maresca, J., Kachar, B., Chadwick, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Elastic+Moduli+of+Thin+Layers+of+Soft+Material+Using+the+Atomic+Force+Microscope.">Determination of Elastic Moduli of Thin Layers of Soft Material Using the Atomic Force Microscope.</a> Biophys J. 82, 2798-2810 (2002).</li><li>Mahaffy, R. E., Shih, C. K., MacKintosh, F. C., K&#228;s, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+Probe-Based+Frequency-Dependent+Microrheology+of+Polymer+Gels+and+Biological+Cells.">Scanning Probe-Based Frequency-Dependent Microrheology of Polymer Gels and Biological Cells.</a> Phys Rev Lett. 85, 880-883 (2000).</li><li>Amin, M., Le, V. P., Wagenseil, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+Testing+of+Mouse+Carotid+Arteries:+from+Newborn+to+Adult.">Mechanical Testing of Mouse Carotid Arteries: from Newborn to Adult.</a> J Vis Exp. , e3733 (2012).</li><li>Laurent, S., 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=Expert+consensus+document+on+arterial+stiffness:+methodological+issues+and+clinical+applications.">Expert consensus document on arterial stiffness: methodological issues and clinical applications.</a> Eur Heart J. 27, 2588-2605 (2006).</li><li>Plodinec, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nanomechanical+signature+of+breast+cancer.">The nanomechanical signature of breast cancer.</a> Nat Nano. 7, 757-765 (2012).</li><li>Raman, A., 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=Mapping+nanomechanical+properties+of+live+cells+using+multi-harmonic+atomic+force+microscopy.">Mapping nanomechanical properties of live cells using multi-harmonic atomic force microscopy.</a> Nat Nano. 6, 809-814 (2011).</li></ol>

The authors have nothing to disclose.

AFMのインデントは、細胞および組織の剛性（弾性率）を特徴付けるために使用することができます。本稿では、マウスで下行大動脈および大動脈弓を隔離し、これらの動脈領域のex vivoでの弾性係数を決定するための詳細なステップバイステップのプロトコルを提供します。私たちは今、要約し、この論文に記載された方法の技術的な問題と制限事項について説明します。 <p class="jo...

The biomechanical properties of arteries are a critical determinant in cardiovascular disease (CVD) and aging. Arterial stiffness, a major cholesterol independent risk factor and an indicator for the progression of CVD, increases with vascular injury, atherosclerosis, age, and diabetes1-8. Arterial wall stiffening is associated with increased dedifferentiation, migration, and proliferation of vascular smooth muscle cells9-12. In addition, increased arterial stiffness has been linked to enhanced macrophage adhesion1, endothelial permeability and leukocyte transmigration13, and vessel wall remodeling14,15. Thus, therapies that could prevent arterial stiffening in CVD or aging might complement currently available pharmacological interventions that treat CVD by reducing high blood cholesterol. 
AFM is a powerful analytical tool used for various physical and biological applications. AFM is increasingly used to obtain the high-resolution images and characterize the biomechanical properties of soft biological samples such as tissues and cells1,2,10,16,17 with a great degree of accuracy at nanoscale levels. A major advantage of AFM is the fact that it can be used with living cells.
This paper describes our method for measuring the elastic modulus of mouse arteries ex vivo using AFM. The described method shows how we 1) properly isolate mouse arteries (descending aorta and aortic arch) and 2) measure the elastic modulus of these tissues by AFM. Measurements of unloaded elastic moduli in arteries can help to elucidate changes in the extracellular matrix (ECM) that occur in response to vascular injury, CVD, and aging.

<table border="0" cellpadding="0" cellspacing="0" width="836">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:272px;">BioScope Catalyst AFM system</td>
			<td style="width:191px;">Bruker</td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nikon Eclipse TE 200 inverted microscope</td>
			<td>Nikon Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Silicon nitride AFM probe</td>
			<td>Novascan Technologies</td>
			<td>PT.SI02.SN.1</td>
			<td>0.06 N/m cantilever; 1 &#181;m SiO2 particle</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dumont #5 forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td>See section 1.4</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dumont #5SF forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td>See section 1.8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Fine Scissors-ToughCut</td>
			<td>Fine Science Tools</td>
			<td style="width:119px;">14058-11</td>
			<td>See section 1.4 (medium sized)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vannas-T&#252;bingen spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15008-08</td>
			<td>See section 1.6 (small sized)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">60mmTC-treated cell culture dish</td>
			<td>Corning</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#39;s Phosphate-Buffered Saline, 1X</td>
			<td>Corning</td>
			<td>21-031-CM</td>
			<td>Without calcium and magnesium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Krazy Glue instant all purpose liquid</td>
			<td>Krazy Glue</td>
			<td>KG58548R</td>
			<td>See section 2.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gel-loading tips, 1-200 &#181;L</td>
			<td>Fisher</td>
			<td>02-707-139</td>
			<td>See section 2.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tip Tweezers</td>
			<td>Electron Microscopy Sciences</td>
			<td>78092-CP</td>
			<td>See section 3.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50-mm, clear wall glass bottom dishes</td>
			<td>TED PELLA</td>
			<td>14027-20</td>
			<td>See section 4.4</td>
		</tr>
	</tbody>
</table>

measuring the stiffness of ex vivo mouse aortas using atomic force microscopy

動脈硬化は、心血管疾患や老化の顕著な特徴のための重要な危険因子とバイオマーカーです。原子間力顕微鏡（AFM）（ 等のプラスチック、ガラス、金属、）ハードに至るまで様々な材料のための粘弾性の機械的性質を特徴付けるための多目的な分析ツールである任意の基板上のセルに表面。広く、細胞の剛性を測定するために使用されるが、あまり頻繁に大動脈の剛性を測定するために使用されてきました。本稿では、無負荷マウスの動脈のex vivoでの弾性率を測定するために、接触モードでAFMを使用するための手順について説明します。我々は、マウスの大動脈の単離のために私たちの手順を説明し、その後、AFM分析のための詳細な情報を提供します。これは、レーザービームのアライメント、ばね定数とAFMプローブの偏向感度のキャリブレーション、および力曲線を取得するためのステップバイステップの手順が含まれています。また、データANALYための詳細なプロトコルを提供します力曲線のsisの。

<html> 図5Aは、6ヶ月齢、雄のC57BL / 6マウスから降順（胸部）大動脈の位相コントラスト画像を示しています。 AFMカンチレバーは、直接組織上記の場所にあり、インデントのための準備ができました。 図5Bおよび5Cは、コンタクトモードでのAFMインデントによって得られた代表的な力曲線を示しています。 図5B及び図5Cに示す緑色の...

Watch this Scientific Journal Video about Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy at JoVE.com

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

の剛性を測定します<em&gt; ex vivoで</em原子間力顕微鏡を使用&gt;マウス大動脈

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

measuring-stiffness-ex-vivo-mouse-aortas-using-atomic-force

Research

JoVE Journal

Biology

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

10.4K ビュー.  University of Pennsylvania. We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

ビデオ: Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Targeted Expression of GFP in the Hair Follicle Using Ex Vivo Viral Transduction

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft during early anagen, the growth phase of the hair cycle, as well as pluripotent stem cells that play a role in hair follicle growth but have the potential to differentiate to non-follicle cells such as neurons. These properties of the hair follicle are discussed. The various cell types of the hair follicle are potential targets for gene therapy. Gene delivery system for the hair follicle using viral vectors or liposomes for gene targeting to the various cell types in the hair follicle and the results obtained are also discussed.

The hair follicle is a highly complex appendage of the skin containing a multiplicity of cell types. The follicle undergoes constant cycling through the life of the organism including growth and resorption with growth dependent on specific stem cells. The targeting of the follicle by genes and stem cells to change its properties, in particular, the nature of the hair shaft is, discussed. Hair follicle delivery systems are described, such as liposomes and viral vectors for gene therapy. The nature of the hair follicle stem cells is discussed, in particular, its pluripotency.

ex vivoでのウイルス形質導入を用いた毛包におけるGFPの標的発現

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft ...

生物学

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

光トラップを用いた細菌細胞の曲げ剛性の測定

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

集積光と原子間力顕微鏡による研究機械的刺激に対する細胞応答を生きる

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

原子間力顕微鏡を用いた肺組織のマイクロメカニカル特性

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

3&#39;UTR -レポーターコンストラクトとマイクロRNAを使用してLightSwitchルシフェラーゼアッセイシステムによるヒトマイクロRNAの識別対象は付着細胞へのミミック

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

小説<em&gt;エクスビボ</emマウス胎児ハート&gt;文化法

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

単一のビリオン原子間力顕微鏡と超解像蛍光イメージングのサンプル調製

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

インフィールドアプリケーションの設計およびアプタマー金ナノ粒子基づく比色アッセイの開発

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

生体共焦点顕微鏡を用いてリンパ節構造と細胞局在化を可視化

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the construction and regeneration of tissues, and is critical in embryonic development, immunological responses, and wound healing. Dysregulation of cell motility contributes to pathological disorders, such as chronic inflammation and cancer metastasis. Cell migration, tissue invasion, axon, and dendrite outgrowth all initiate with actin polymerization-mediated cell-edge protrusions. Here, we describe a simple, efficient, time-saving method for the imaging and quantitative analysis of cell-edge protrusion dynamics during spreading. This method measures discrete features of cell-edge membrane dynamics, such as protrusions, retractions, and ruffles, and can be used to assess how manipulations of key actin regulators impact cell-edge protrusions in diverse contexts.

This protocol aims to measure the dynamic parameters (protrusions, retractions, ruffles) of protrusions at the edge of spreading cells.

生細胞顕微鏡による拡散時の細胞端突出ダイナミクスの測定

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the ...