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>
	<li>Kothapalli, D., 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=Cardiovascular+Protection+by+ApoE+and+ApoE-HDL+Linked+to+Suppression+of+ECM+Gene+Expression+and+Arterial+Stiffening.">Cardiovascular Protection by ApoE and ApoE-HDL Linked to Suppression of ECM Gene Expression and Arterial Stiffening.</a> Cell Rep. 2, 1259-1271 (2012).</li><li>Liu, S. -. L., 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=Matrix+metalloproteinase-12+is+an+essential+mediator+of+acute+and+chronic+arterial+stiffening.">Matrix metalloproteinase-12 is an essential mediator of acute and chronic arterial stiffening.</a> Sci Rep. 5, 17189 (2015).</li><li>Lakatta, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+arterial+aging+and+the+epidemic+of+systolic+hypertension+and+atherosclerosis.">Central arterial aging and the epidemic of systolic hypertension and atherosclerosis.</a> J Am Soc Hypertens. 1, 302-340 (2007).</li><li>Stehouwer, C. D. A., Henry, R. M. A., Ferreira, I. <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+in+diabetes+and+the+metabolic+syndrome:+a+pathway+to+cardiovascular+disease.">Arterial stiffness in diabetes and the metabolic syndrome: a pathway to cardiovascular disease.</a> Diabetologia. 51, 527-539 (2008).</li><li>Steppan, J., Barodka, V., Berkowitz, D. E., Nyhan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+Stiffness+and+Increased+Pulse+Pressure+in+the+Aging+Cardiovascular+System.">Vascular Stiffness and Increased Pulse Pressure in the Aging Cardiovascular System.</a> Cardiol Res Pract. 2011, 263585 (2011).</li><li>Duprez, D. A., Cohn, J. N. <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+as+a+risk+factor+for+coronary+atherosclerosis.">Arterial stiffness as a risk factor for coronary atherosclerosis.</a> Curr Atheroscler Rep. 9, 139-144 (2007).</li><li>Mitchell, G. 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 indentazione può essere utilizzato per caratterizzare la rigidità (modulo elastico) di cellule e tessuti. In questo lavoro, forniamo dettagliate protocolli passo-passo per isolare l&#39;aorta discendente e arco aortico nel topo e determinare il modulo elastico di queste regioni arteriosi ex vivo. Ora riassumere e discutere le questioni tecniche e le limitazioni del metodo descritto in questo articolo. Diversi problemi tecnici possono sorgere in isolamento e l&#39;analisi di topo aorte dato il loro piccolo e sottile della natura. Quando le arterie pulite vengono aperte longitudinalmente, bisogna fare attenzione a non disturbare il intima (superficie luminale) in cui vengono raccolti i dati modulo elastico. Eccesso PBS nel piatto finale campione impedisce il corretto incollaggio delle arterie sul piatto, e questo deve essere rimosso accuratamente con un fazzoletto di carta senza toccare l&#39;arteria prima dell&#39;incollaggio. Si noti, tuttavia, che l&#39;arteria isolata può aderire alla WHI tessuto di cartaLe tentare di rimuovere eccessivo PBS, e questo può danneggiare il campione. Mentre l&#39;incollaggio delle arterie al piatto di coltura, è estremamente importante che l&#39;arteria non viene allungato e che soltanto una piccola quantità di adesivo viene applicato bit per bit a ciascuna estremità dell&#39;arteria. Quando si esegue l&#39;AFM-indentazione, è fondamentale per evitare sondare alle estremità dell&#39;arteria in cui è stato applicato l&#39;adesivo. Infine, perché arterie appena isolate sono utilizzati per le misure di modulo elastico, il tempo trascorso dalla raccolta misura deve essere minimizzata. Poiché le punte sferiche utilizzati in questo studio hanno un raggio di 500 nm, una profondità di penetrazione di fino a 500 nm può essere accuratamente analizzati utilizzando il modello Hertz. Questa profondità indentazione sarebbe correlata con lo strato superiore delle cellule endoteliali situati nell&#39;intima 21. Per sondare la meccanica degli strati sub-endoteliale dei campioni arteriosi, sonde colloidali più grandi (10 - 20 micron di diametro) potrebbe essere utilizzatoper l&#39;indentazione e cambiamenti nella rigidità calcolato con profondità di penetrazione è stato possibile determinare 22,23. Inoltre, poiché i campioni biologici sono solitamente viscoelastico non puramente elastico, misurazioni di stress rilassamento (monitoraggio decadimento in forza applicata ad una profondità di indentazione costante) possono essere utilizzati per determinare la componente viscosa arteriose proprietà meccaniche 24,25. Un potenziale limitazione di analisi AFM è la dimensione di scansione, che misura solamente una piccola regione di tessuto. La maggior parte dei campioni biologici, comprese le arterie e le cellule, non sono topograficamente e biomeccanicamente omogenea, e questa eterogeneità può potenzialmente causare artifactually grandi variazioni del modulo elastico a seconda scorrevolezza del sito indentazione, profondità di indentazione, e la quantità di forza applicata al campione superficie. Per ottenere un valore rappresentativo medio del modulo elastico generale, è importante effettuare ripetute AFM-indentazione unt più posizioni casuali attraverso le arterie, come mostrato in figura 4. Inoltre, si usa una punta sferica di dimensioni adeguate, come studi precedenti hanno dimostrato che AFM-indentazione con una punta piramidale affilata determinato maggiori stime del modulo elastico e danneggiato morbido campioni biologici 26,27. Anche se i nostri ex vivo misure AFM di rigidità arteriosa sembrano concordare bene con marcatori molecolari di cellule muscolari lisce irrigidimento 2, è importante rendersi conto che questi campioni arteriosi non vengono caricati meccanicamente e carico meccanico è probabile che a giocare un ruolo importante nella complessiva meccanica arteriose. I confronti di rigidità arteriosa come determinato da AFM, myography (un metodo ex vivo compatibile con carico meccanico) 28 e la velocità degli impulsi-onda (una misurazione in vivo della rigidità arteriosa) 29 probabilmente fornirà la comprensione più completa di Artermeccanica IAL. Abbiamo descritto l&#39;AFM in modalità contatto per misurare la ex vivo rigidità delle arterie del mouse. Questo metodo stima la rigidità media dei tessuti per il rientro in modo casuale su più regioni dell&#39;aorta. Per esaminare la distribuzione spaziale della rigidità delle regioni più grandi di tessuti, un recente lavoro ha utilizzato AFM in modalità forza del volume 30. Questo approccio contemporaneamente ottenuta una mappa di variazioni di rigidezza ed una immagine ad alta risoluzione totali topografica del tessuto. Oltre a misurare la rigidità di campioni di tessuto, la AFM in modalità forza volume può essere utilizzato in celle per monitorare le distribuzioni spaziali di rigidità intracellulare 10,31.

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

irrigidimento arteriosa è un fattore di rischio significativo e biomarker per malattie cardiovascolari e un segno distintivo di invecchiamento. Microscopia a forza atomica (AFM) è uno strumento analitico versatile per la caratterizzazione viscoelastiche proprietà meccaniche per una varietà di materiali che vanno dal disco (plastica, vetro, metallo, ecc) le superfici di celle su qualsiasi substrato. È stato ampiamente utilizzato per misurare la rigidità delle cellule, ma usato meno frequentemente per misurare la rigidità di aorte. In questo articolo, descriveremo le procedure per l&#39;utilizzo in modalità AFM contatto per misurare la ex vivo modulo elastico delle arterie scaricati mouse. Descriviamo la nostra procedura per l&#39;isolamento di aorte del mouse, e quindi fornendo informazioni dettagliate per l&#39;analisi AFM. Questo include le istruzioni passo-passo per l&#39;allineamento del raggio laser, la taratura della costante della molla e la sensibilità deflessione della sonda AFM, e l&#39;acquisizione di curve di forza. Forniamo anche un protocollo dettagliato per analy datisis delle curve di forza.

<html> La figura 5A mostra un&#39;immagine a contrasto di fase dell&#39;aorta discendente (toracica) da un 6 mesi di età, di sesso maschile C57BL / 6 del mouse. Il cantilever AFM è posto direttamente sopra il tessuto e pronto per il rientro. Figure 5B e 5C mostrano curve rappresentative forza ottenuti mediante AFM rientranza in modalità contatto. Linee verdi mostrate nelle figure 5B e 5C rappresentano le migliori curve di adattamento ottenuti utilizzando il modello di Hertz per una sfera. Nella Figura 5D, la rigidità media delle aorte discendente e archi aortici è determinata da tre topi individuo come descritto nel testo. Si noti che l&#39;aorta discendente e l&#39;arco aortico rappresentano regioni di flusso laminare e disturbato, rispettivamente. Tuttavia, le loro (scaricati) meccanicamente moduli elastici sono simili come determinato mediante AFM. 0 / 54630fig5.jpg &quot;/&gt; Figura 5: Le analizzati curve forza-indentazione e la rigidità microfotografia dati (a) contrasto di fase che mostra un cantilever AFM su un mouse aorta discendente ex vivo.. Barra della scala, 100 micron. (BC) Un insieme di due curve rappresentative forza-indentazione acquistati per (B) l&#39;aorta discendente e (C) aortico. (D) Media rigidità dell&#39;aorta del mouse discendente e arco aortico è stato determinato come descritto sopra. Le barre di errore mostrano media + SEM; n = 3 repliche biologiche indipendenti. <a href="https://www.jove.com/files/ftp_upload/54630/54630fig5large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a>

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.

Misurare la rigidità<em&gt; Ex Vivo</em&gt; Mouse aorte Utilizzando microscopia a forza atomica

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

Nanyang Technological University

Research

JoVE Journal

Biology

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

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

Video: 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.

Espressione mirato di GFP nel follicolo pilifero Utilizzando Ex trasduzione virale Vivo

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 ...

Ricerca

Biologia

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.

Misurare la rigidezza flessionale di cellule batteriche Utilizzando una trappola ottica

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.

Risposta cellulare allo stimolo meccanico vivere Studiato da Integrated ottica e microscopia a forza atomica

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.

Micro-Caratterizzazione meccanica del tessuto polmonare usando la microscopia a forza atomica

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.

Obiettivi Identificazione di microRNA umani con il sistema di analisi Luciferasi LightSwitch utilizzando 3&#39;UTR-reporter Costruisce e un Mimic microRNA in cellule aderenti

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.

A Novel<em&gt; Ex vivo</em&gt; Metodo di Cultura per il mouse embrionale del cuore

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.

Preparazione del campione per microscopia a forza atomica Virion singola e imaging a fluorescenza a super risoluzione

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

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.

Visualizzazione della struttura dei linfonodi e della localizzazione cellulare mediante la microscopia confocale Ex-Vivo

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.

Misurazione della dinamica di protrusione del bordo cellulare durante la diffusione utilizzando la microscopia a cellule vive

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

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young&#39;s modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

Caratterizzazione delle proprietà meccaniche della parete cellulare primaria in organi vegetali viventi mediante microscopia a forza atomica

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the ...