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.

כניסת AFM יכול לשמש כדי לאפיין את הנוקשות (מודולוס אלסטיות) של תאים ורקמות. במאמר זה, אנו מספקים פרוטוקולים צעד-אחר-צעד מפורט לבודד את אבי העורקים היורד ואת קשת אבי העורקים בתוך העכבר ולקבוע את moduli אלסטי של אזורים עורקים אלה vivo לשעבר. אנחנו עכשיו לסכם ולדון בסוגיות המגבלות הטכניות של השיטה המתוארת במאמר זה. כמה בעיות טכניות עלולות להתעורר בבידוד והניתוח של aortas עכבר בהתחשב באופי הקטנה ורזה שלהם. כאשר העורקים ניקו נפתחים אורכים, יש להקפיד לא לשבש את אינטימה (המשטח לומינל) שבו הנתונים מודולוס אלסטיות נאספות. עודף PBS בצלחת המדגם הסופי מונעת הדבקה נאותה של העורקים על הצלחת, וזה צריך להיות מוסר בזהירות בעזרת ממחטת נייר בלי לגעת העורק לפני ההדבקה. שים לב, עם זאת, כי העורק המבודד יכול להיצמד WHI ממחטת ניירle להסרתה PBS מופרז, ודבר זה יכול לגרום נזק המדגם. בעוד הדבקת העורקים אל מנת התרבות, חשוב מאוד כי העורק אינו נמתח וכי כמות של דבק קטן מוחלת קצת-ידי סיבי לכל קצה של העורק. בעת ביצוע-הזחה AFM, היא קריטית כדי למנוע חיטוט בקצות העורק שבו דבק יושם. לבסוף, כי עורקים מבודדים טרי משמשים את המידות של מודולוס אלסטיות, הזמן שחלף מן הקציר מדידה צריך להיות ממוזער. מאז טיפים הכדוריים המשמשים במחקר זה יש ברדיוס של 500 ננומטר, עומק הזחה של עד 500 ננומטר ניתן לנתח במדויק באמצעות מודל ההרץ. עומק הזחה זה היה לתאם עם השכבה העליונה של תאי האנדותל ממוקמים אינטימה 21. כדי לחקור את המכניקה של שכבות תת-אנדותל של דגימות עורקים, בדיקות קולואידים גדול (10 - 20 מיקרומטר קוטר) יכול לשמשעבור כניסה ושינויים קשיחים מחושב עם עומק כניסה ניתן לקבוע 22,23. כמו כן, מאז דגימות ביולוגיות הן בדרך כלל viscoelastic ולא גרידא אלסטי, מדידות הרפית מתח (ניטור הדעיכה בתוקף יישומים בעומק זחה קבוע) יכולות לשמש כדי לקבוע את המרכיב הצמיג של תכונות מכאניות עורקי 24,25. מגבלה פוטנציאל של ניתוח AFM היא גודל הסריקה, אשר מודד רק אזור קטן של הרקמה. רוב דגימות ביולוגיות, כולל העורקים התאים, הם לא טופוגרפית biomechanically הומוגנית, ההטרוגניות זה יכול לגרום באופן פוטנציאלי שינויים גדולים artifactually של מודולוס אלסטיות תלוי החלקות של אתר הכניסה, עומק הכניסה, ואת כמות הכוח המופעל על המדגם משטח. כדי לקבל ערך נציג ממוצע של מודולוס הכוללת אלסטי, חשוב לבצע AFM-זחה חזרt מספר מיקומים אקראיים דרך העורקים, כפי שמוצגים באיור 4. יתר על כן, אנו משתמשים קצה כדורי בגודל מתאים, כמו מחקרים קודמים הראו כי AFM-זח עם קצה פירמידה חד הביאו אומדנים גבוהים יותר של מודולוס האלסטיות ופגעתי רך דגימות ביולוגיות 26,27. למרות מדידות AFM vivo לשעבר שלנו של קשיות עורקים מופיעות להסכים גם עם סמנים מולקולריים של תאי שריר חלק מקשיח 2, חשוב להבין כי דגימות העורקות אלה אינן נטענות מכאני, ואת העומס מכאני עשוי לשחק תפקיד חשוב כללי מכניקה עורקת. השוואות של קשיות העורקים כפי שנקבע על ידי AFM, myography (שיטה vivo לשעבר תואם טעינה מכני) 28 ומהירות הדופק-גל (מדידה in vivo של קשיות העורקים) 29 צפוי לספק את ההבנה המקיפה ביותר של arterמכניקת ial. תארנו את AFM במצב מגע, המודד את נוקשות vivo לשעבר של עורקי עכבר. שיטה זו מעריכה את רקמות נוקשות הממוצע ע&quot;י הפנמה באקראי על אזורים מרובים של אבי העורקים. כדי לבחון את הפריסה המרחבית של קשיחות מאזורים גדולים של רקמות, נייר אחרון השתמש AFM במצב כוח בנפח 30. גישה זו זמנית השיגה מפה של וריאציות קשיחות תמונה ברזולוציה גבוהה של פני שטח טופוגרפיים של הרקמה. בנוסף למדידת קשיות של דגימות רקמה, ה- AFM במצב כוח בנפח ניתן להשתמש בתאים לפקח על הפצות המרחבי של נוקשות תאיים 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

ההתקשות עורקת היא גורם סיכון משמעותי סמן למחלות לב וכלי דם סימן היכר של הזדקנות. מיקרוסקופ כוח אטומי (AFM) הוא כלי אנליטי צדדי לאפיון תכונות מכאניות viscoelastic עבור מגוון רחב של חומרים, החל קשה (פלסטיק, זכוכית, מתכת, וכו &#39;) משטחים לתאים על מצע כלשהו. זה כבר נעשה שימוש נרחב על מנת למדוד את הנוקשות של תאים, אך בתדירות נמוכה יותר משמש כדי למדוד את הנוקשות של aortas. במאמר זה, נתאר את ההליכים באמצעות AFM במצב מגע, מודדי מודולוס vivo לשעבר אלסטי של עורקי עכבר טעונים. אנו מתארים הליך שלנו לבידוד תרמי של aortas עכבר, ולאחר מכן לספק מידע מפורט לניתוח AFM. זה כולל הוראות שלב-אחר-שלב עבור יישור של קרן לייזר, כיול של קבוע הקפיץ ורגישות סטיה של החללית AFM, ורכישת עקומות כוח. כמו כן אנו מספקים פרוטוקול מפורט analy נתוניםאחותי של עקומות כוח.

<html> איור 5 א מראה תמונה בניגוד שלב של אב העורקים היורד (חזה) מילדה בת 6 חודשים, עכבר C57BL / 6 גברים. שלוחת AFM היא במקום ישירות מעל הרקמה ומוכנה זחה. 5B הדמוי ו -5 ג להפגין עקומות כוח נציג מתקבלות על ידי זחת AFM במצב מגע. קווים ירוקים שמוצגים איורי 5 ב ו -5 ג לייצג את העקומות בכושר הטובות ביותר המתקבלות כתוצאה מהפעלת מודל Hertzian עבור כדור. באיור 5D, הנוקשות הממוצעות של aortas יורד וקשתות אב עורקים נקבעו משלושה עכברים בודדים כמתוארות בטקסט. ראוי לציין, כי אבי העורקים היורד ואת קשת אבי העורקים מייצגים אזורים של למינרית וזרימה מופרע, בהתאמה. אף על פי כן, שלהם (פרקו המכאני) moduli אלסטי דומה כפי שנקבע על ידי AFM. 0 / 54630fig5.jpg &quot;/&gt; איור 5: Curves חיל-הזחה המנותחת נוקשות נתונים (א) מיקרוסקופ בניגוד שלב מציג שלוחת AFM מעל עכבר יורד האאורטה vivo לשעבר.. סרגל קנה מידה, 100 מיקרומטר. (בי סי) סט של שתי עקומות כוח-הזחה נציג רכשה עבור (B) אבי העורקים היורד ו- (ג) קשת אבי העורקים. (ד) Mean נוקשות של אבי העורקים היורד העכבר קשת אבי העורקים נקבע על פי הפירוט מתואר לעיל. ברים שגיאים להראות ממוצעת + SEM; n = 3 משכפל ביולוגי עצמאי. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54630/54630fig5large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</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.

מדידת הקשיחות של<em&gt; Ex Vivo</em&gt; עכבר Aortas באמצעות מיקרוסקופ כוח אטומי

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.

ביטוי ממוקד של ה-GFP ב זקיק השיער באמצעות אקס התמרה Vivo ויראלי

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 ...

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.

מדידת קשיחות כיפוף של תאים חיידקיים באמצעות מלכודת אופטית

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.

תגובת תא חי כדי גירוי מכני שנלמדה על ידי מיקרוסקופית כוח משולב אופטי אטומית

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.

מיקרו מכונות אפיון רקמת הריאה באמצעות מיקרוסקופ כוח אטומי

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.

זיהוי מטרות של רנ&quot;א האדם עם מערכת Assay בלוציפראז LightSwitch באמצעות 3&#39;UTR-הכתב בונה microRNA מחקים בתאים חסיד

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.

רומן<em&gt; Ex vivo</em&gt; שיטת תרבות ללב העכבר העוברית

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.

הכנה לדוגמה למיקרוסקופיה של כוח אטומי של Virion יחיד והדמיית פלואורסצנטיות ברזולוציה גבוהה במיוחד

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.

עיצוב ופיתוח של מבוסס Nanoparticle Aptamer-זהב מדד-צבע מבחנים עבור יישומי in-the-השדה

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.

חזותי מבנה צומת לימפה ולוקליזציה לסלולר באמצעות Ex-Vivo Confocal מיקרוסקופ

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.

מדידת דינמיקת בליטה בקצה התא במהלך ההתפשטות באמצעות מיקרוסקופיה של תאים חיים

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