Name: studying the effects of matrix stiffness on cellular function using acrylamide-based hydrogels
Uploaded: 2010-08-10T17:00:00
Description: Watch this Scientific Journal Video about Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels at JoVE.com

İş bizim laboratuar Ulusal Sağlık Enstitüleri hibe tarafından desteklenmektedir.

Hazırlık <ul><li> Lameller otoklava olmalıdır. </li><li> Steril distile veya deiyonize su çözümler hazırlamak ve çamaşır lamelleri için kullanılmalıdır. </li><li> AC (% 40 w / v) ve bis-AC (% 1 w / v) çözümleri 0.2 mikron filtre ile sterilize edilir. Kullanımı ve steril filtre kısa bir süre önce,% 10 amonyum persülfat (100μg/ml su APS) hazırlayın. APS çözüm aylık değiştirin. </li><li> 3-APTMS, kloroform, glutaradehyde, NHS ve SurfaSil gibi kimyasal reaktifler otoklavlanmalıdır sadece...

<ol>
	<li>Beattie, D., Xu, C., Vito, R., Glagov, S., Whang, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+analysis+of+heterogeneous,+atherosclerotic+human+aorta.">Mechanical analysis of heterogeneous, atherosclerotic human aorta.</a> J Biomech Eng. 120, 602-607 (1998).</li><li>Bernini, G. <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,+intima-media+thickness+and+carotid+artery+fibrosis+in+patients+with+primary+aldosteronism.">Arterial stiffness, intima-media thickness and carotid artery fibrosis in patients with primary aldosteronism.</a> J Hypertens. 26, 2399-2405 (2008).</li><li>Boonyasirinant, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortic+stiffness+is+increased+in+hypertrophic+cardiomyopathy+with+myocardial+fibrosis:+novel+insights+in+vascular+function+from+magnetic+resonance+imaging.">Aortic stiffness is increased in hypertrophic cardiomyopathy with myocardial fibrosis: novel insights in vascular function from magnetic resonance imaging.</a> J Am Coll Cardiol. 54, 255-2562 (2009).</li><li>Discher, D. E., Janmey, P., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+cells+feel+and+respond+to+the+stiffness+of+their+substrate.">Tissue cells feel and respond to the stiffness of their substrate.</a> Science. 310, 1139-1143 (2005).</li><li>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>Lee, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+mechanical+properties+of+human+atherosclerotic+tissue+by+high-frequency+intravascular+ultrasound+imaging.+An+in+vitro+study.">Prediction of mechanical properties of human atherosclerotic tissue by high-frequency intravascular ultrasound imaging. An in vitro study.</a> Arterioscler Thromb. 12, 1-5 (1992).</li><li>Levental, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+crosslinking+forces+tumor+progression+by+enhancing+integrin+signaling.">Matrix crosslinking forces tumor progression by enhancing integrin signaling.</a> Cell. 139, 891-906 (2009).</li><li>Paszek, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensional+homeostasis+and+the+malignant+phenotype.">Tensional homeostasis and the malignant phenotype.</a> Cancer Cell. 8, 241-254 (2005).</li><li>Samani, A., Zubovits, J., Plewes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+moduli+of+normal+and+pathological+human+breast+tissues:+an+inversion-technique-based+investigation+of+169+samples.">Elastic moduli of normal and pathological human breast tissues: an inversion-technique-based investigation of 169 samples.</a> Phys Med Biol. 52, 1565-1576 (2007).</li><li>Wells, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+matrix+stiffness+in+regulating+cell+behavior.">The role of matrix stiffness in regulating cell behavior.</a> Hepatology. 47, 1394-1400 (2008).</li><li>Pelham, R. J., Wang, Y. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+locomotion+and+focal+adhesions+are+regulated+by+substrate+flexibility.">Cell locomotion and focal adhesions are regulated by substrate flexibility.</a> Proc. Natl. Acad Sci USA. 94, 13661-13665 (1997).</li><li>Klein, E. A., Yung, Y., Castagnino, P., Kothapalli, D., Assoian, R. 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Hidrojel polimerizasyon işlemi çok önemli bir unsur hücreleri ECM-kaplı hidrojel kendini daha ziyade cam lamel bağlamak sağlayacak hava kabarcığı oluşumunu önlemek için. Bu vorteks ve görsel hiçbir hava kabarcığı jel tuzak haline emin sonra dikkatli bir şekilde polimerizasyon çözüm pipetleme önlenebilir. Biz her zaman deneyler için yeterince sağlamak için ek &quot;reaktif&quot; lamelleri ve hidrojellerin hazırlanması önerilir. Jeller PBS ile yıkanır zaman özel...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Glutaraldehyde, 70%</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G7776</td>
<td>Store at -20&deg;C</td>
</tr>
<tr>
<td>3-APTMS (3-Aminopropyltrimethosysilane 97%)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>281778</td>
<td>Store at room temperature</td>
</tr>
<tr>
<td>SurfaSil Siliconizing Fluid</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>42800</td>
<td>Store at room temperature</td>
</tr>
<tr>
<td>NHS (N-hydroxysucinimide Ester)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A-8060</td>
<td>Store at 4&deg;C Replace monthly</td>
</tr>
<tr>
<td>Albumin, bovine serum, essentially fatty acid free</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A6003-100G</td>
<td>Store at 4&deg;C</td>
</tr>
<tr>
<td>Coverslips (25mm)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-86 25 Cir 1D</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips (18mm)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-84 18 Cir 1D</td>
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studying the effects of matrix stiffness on cellular function using acrylamide-based hydrogels

Doku sertliği hücresel fonksiyonunun önemli bir belirleyicisi olup, doku sertliği değişiklikler yaygın fibrozis, kanser ve kardiyovasküler hastalık 1-11 ile ilişkilidir. Geleneksel hücre biyolojik yaklaşımlar hücresel fonksiyonu çalışmalarında elastik bir ECM ya da dokular arasında ECM sertlik değişikliklerin etkisini dikkate almaz, katı bir alt tabaka üzerinde kültür hücreleri (plastik tabaklar ya da cam lamelleri) içerir. In vitro, in vivo doku uyumu şartları model için, biz ve diğerleri ECM-kaplı hidrojeller kullanın. Laboratuvarımızda, hidrojeller dizi 12 biyolojik olarak görülen doku uygunluk bildirimleri taklit edebilir poliakrilamid dayanmaktadır. &quot;Reaktif&quot; kapak fişleri 3-APTMS Ayrıca NaOH ile kuluçka tarafından üretilir. Glutaraldehyde 3-APTMS ve poliakrilamid jel çapraz bağlamak için kullanılır. Akrilamid (AC), bis-akrilamid (Bis-AC) ve amonyum persülfat bir çözüm hidrojel polimerizasyon için kullanılır. N-hydroxysuccinimide (NHS), hidrojel çapraz ECM protein AC çözüm içine dahil edilmiştir. Hidrojel polimerizasyonu sonra jel yüzeyi, fibronektin, vitronectin, kollajen, vb gibi bir seçim ECM protein ile kaplıdır Bir hidrojel sertlik reoloji veya atomik kuvvet mikroskobu (AFM) tarafından belirlenir ve çözüm 12 AC ve / veya bis-AC yüzdesi değişen ayarlanabilir. Bu şekilde, alt tabaka sertlik reoloji veya AFM kullanılarak belirlenebilir Biyolojik dokuların sertlik eşleştirilmiş olabilir. Hidrojeller ve kültürlü gerekli deneysel koşullar dayalı bu hücreler daha sonra numaralı seribaşı olabilir. Moleküler analiz için hücre ve onların kurtarma Görüntüleme basittir. Bu yazı için, yumuşak katmanlarından 20.000 Pascal &lt;E 3000 Pascal ve sert doku katmanlarından /&gt; elastik modülü (E) olarak tanımlar.

Watch this Scientific Journal Video about Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels at JoVE.com

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Hücresel fonksiyonları katmanlarından sertlik etkisi modellenmiş olabilir<em&gt; In vitro</em&gt; Uyumluluklar değişen poliakrilamid hidrojeller.

Matrix Rijitlik Akrilamid tabanlı Hidrojeller kullanarak Hücresel Fonksiyon Üzerindeki Etkilerinin incelenmesi

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

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