We are grateful to the Roney Family Foundation for the support of this study. We acknowledge the contribution of Dr. M. Lucrecia Alvarez to the planning and execution of the experiments described in this work.

Not:. Modifiye çökeltme yöntemi kullanılarak idrar eksozomlann izole söz konusu olan adımlar, Şekil 1 &#39;de gösterilmektedir, ilk adım santrifüj ile büyük ölü hücrelerin çıkarılması ve hücre artıkları içerir. Her bir sonraki aşamada toplanan kültür bakiyesinden elde edilen nihai pelet, protein, RNA ve DNA daha fazla ekstre için ayırma çözeltisi içinde eritilir eksozom tekabül eder. İdrar 1. Koleksiyonu <ol><li> Idrar toplama öncesinde, üreticinin talimatlarına uygun olarak bir proteaz inhibitörü kokteyli hazırlamak ve boş steril hazne Bu kokteyl 3.125 ilave edin. proteaz inhibitörü kokteyli içeren Tüp daha sonra idrar toplama gönüllülere dağıtılabilir. Not: proteaz inhibitörü ilave nedeni idrar numunesi içinde bulunan proteini bozulmasını en aza indirmektir. Biz özellikle bir proteaz inhibitörü kokteyl kullanılmasına rağmenSigma, diğer ticari olarak temin edilebilen proteaz inhibitörleri ikame edilebilir. </li><li> İlk boşluk idrar yaklaşık 10 ml toplayın. Toplandıktan hemen sonra depolama veya soğutma olmadan idrar işleyin. Modifiye yağış yöntemini kullanarak kopya üriner eksozomlar yalıtmak; üriner eksozom partiküllerinin ölçülmesi için ise çiftleri biri, DNA, toplam RNA ve protein ekstre içindir. NOT: 24 saat idrar numunesi 4 ° C&#39;de saklayarak eksozom izolasyonu için toplanabilir. </li></ol> Hücre Moloz ve Tamm-Horsfall Protein 2. Kaldırma (THP) <ol><li> 15 ml santrifüj tüplerine idrar örnekleri aktarın. Santrifüj 37 ° C&#39;de 10 dakika boyunca 17,000 x g&#39;de idrar 10 mi hücreleri ve hücre kalıntısını (Şekil 1) ortadan kaldırmak için. </li><li> Bir pipet kullanarak, daha fazla işlem için yeni bir tüpe süpernatantı aktarın. Izolasyon çözeltisi 500 ul pelletini (250 mM sakaroz, 10 mM Trietilamininnihai konsantrasyon 200 mg / ml), 37 ° C &#39;de DL-ditiyotreitol (DTT ile 10 dakika inkübasyon ile takip anolamine, pH 7.6). Not: DTT verim azalmasına yol açan, eksozomlar yakalar THP, indirgeme eklenir. Süpernatant transferi sırasında rahatsız pelet bırakmak özen, ve yüzer pelet açık olduğunu doğrulayın. </li><li> Vortex pelet örnekleri pelet kadar her 2 dk tamamen çözülür. Ardından çözünmüş pelet izolasyon çözeltisi 500 ul ekleyin. </li><li> 37 ° C, 10 dakika için 17,000 x g&#39;de izole pelet çözeltisi santrifüj ilave edildikten sonra. Pipetleyin süpernatant ve önceki toplanan süpernatant ile birleştirmek. </li><li> SDS-PAGE analizi için ayırma çözeltisi 50 ul pelletini. Not: PBS veya TBS pelletini yerine yalıtım çözeltisi kullanılabilir. </li></ol> Exosome Peleti 3. İzolasyonu <ol><li> Toplanan supern 11 mlatant, ExoQuick-TC ayıracı 3.3 ml ilave edilir ve en az 12 saat süre ile 4 ° C&#39;de inkübe edin. Not: ExoQuick-TC reaktif süpernatant hacmine oranı arttırmak veya exosomal verim azaltmak için ayarlanabilir. Bu ilave reaktif az miktarlar eksozom küçük anomerin görülmektedir. </li><li> Inkübasyondan sonra, santrifüj 25 ° C&#39;de 30 dakika boyunca 10,000 x g&#39;de örnek / ExoQuick-TC karışımı. </li><li> SDS-PAGE analizi için temiz bir tüpe süpernatant aktarın. </li><li> eksozom pelet, DNA, RNA ve protein ekstraksiyonu ve CD9 ELISA 11 kullanılarak eksozom partiküllerinin ölçümü için, -80 ° C&#39;de saklanabilir. Not: eksozom pelet sınırlı dondurma ve eritme ile 1 yıl boyunca, -80 ° C&#39;de saklanabilir. </li></ol> 4. Biyomarker Algılama ve Exosome Peleti Analizi <ol><li> Alt baş analizler için, DNA / RNA / protein izolasyon kiti ve bir RNA izolasyon Ki ile eksozom topağından DNA, RNA ve protein özüüreticinin talimatlarına uygun olarak T. Örnek 2 ul 260 absorbans ve 280nm ölçerek Nanodrop spektrofotometre kullanılarak konsantrasyonlarını belirleyin. </li><li> Kantitatif gerçek zamanlı RT-PCR (qPCR), TaqMan microRNA deneyleri kullanılarak veya miRNA-spesifik primerler (bizim durumumuzda, biz insan miR-192- ve miR-1207-5p özel deneyleri kullanılır) gerçekleştirin. </li><li> SDS-PAGE analizi için, tek boyutlu elektroforez ile% 4-12 Bis-Tris jel ve ayrı protein numuneleri kullanmaktadır. Üreticinin talimatlarına uygun olarak, gümüş boya kitini kullanarak jeller görselleştirmek. </li><li> Üretici tarafından sağlanan talimatlara uygun olarak PVDF membranlar kullanılarak Western blot analizi. Western blot algılama tümör bir gen 101 protein veya TSG101 apoptoz bağlantılı gen-2 etkileşimli protein X veya Alix ve fare monoklonal antikor tavşan poliklonal antikor kullanın. Açık erişim NIH ImageJ yazılımı tarafından bantların yoğunluğu ölçmek (http://rsbweb.nih.gov/ij/) veyabenzer bir program. </li><li> Üreticinin talimatlarına göre bir CD9 ELISA kiti kullanılarak elde edilen idrar eksozom parçacıkların sayısını ölçmek. </li></ol>

<ol>
	<li>Miranda, K. C., Bond, D. T., McKee, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleic+acids+within+urinary+exosomes/microvesicles+are+potential+biomarkers+for+renal+disease.">Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease.</a> Kidney Int. 78, 191-199 (2010).</li><li>Pisitkun, T., Shen, R. F., Knepper, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+proteomic+profiling+of+exosomes+in+human+urine.">Identification and proteomic profiling of exosomes in human urine.</a> Proc Natl Acad Sci USA. 101, 13368-13373 (2004).</li><li>Raj, D. A., Fiume, I., Capasso, G., Pocsfalvi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multiplex+quantitative+proteomics+strategy+for+protein+biomarker+studies+in+urinary+exosomes.">A multiplex quantitative proteomics strategy for protein biomarker studies in urinary exosomes.</a> Kidney Int. 81 (12), 1263-1273 (2012).</li><li>Pisitkun, T., Gandolfo, M. T., Das, S., Knepper, M. A., Bagnasco, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+systems+biology+principles+to+protein+biomarker+discovery:+urinary+exosomal+proteome+in+renal+transplantation.">Application of systems biology principles to protein biomarker discovery: urinary exosomal proteome in renal transplantation.</a> Proteomics Clin Appl. 6 (5-6), 268-278 (2012).</li><li>Gonzales, P. A., Zhou, H., Pisitkun, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+purification+of+exosomes+in+urine.">Isolation and purification of exosomes in urine.</a> Methods Mol Biol. 641, 89-99 (2010).</li><li>Zhou, H., Yuen, P. S., Pisitkun, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collection,+storage,+preservation,+and+normalization+of+human+urinary+exosomes+for+biomarker+discovery.">Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery.</a> Kidney Int. 69, 1471-1476 (2006).</li><li>Thery, C., Amigorena, S., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+exosomes+from+cell+culture+supernatants+and+biological+fluids.">Isolation and characterization of exosomes from cell culture supernatants and biological fluids.</a> Curr Prot Cell Biol. 43, 3.22.1-3.22.29 (2006).</li><li>Cheruvanky, A., Zhou, H., Pisitkun, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+urinary+exosomal+biomarkers+using+a+nanomembrane+ultrafiltration+concentrator.">Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator.</a> Am J Physiol Renal Physiol. 292, 1657-1661 (2007).</li><li>Gonzales, P. A., Pisitkun, T., Hoffert, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+proteomics+and+phosphoproteomics+of+urinary+exosomes.">Large-scale proteomics and phosphoproteomics of urinary exosomes.</a> J Am Soc Nephrol. 20, 363-379 (2009).</li><li>Rood, I. M., Deegens, J. K., Merchant, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+three+methods+for+isolation+of+urinary+microvesicles+to+identify+biomarkers+of+nephrotic+syndrome.">Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome.</a> Kidney Int. 78, 810-816 (2010).</li><li>Alvarez, M. L., Khosroheidari, M., Kanchi Ravi, R., DiStefano, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+protein,+microRNA,+and+mRNA+yields+using+different+methods+of+urinary+exosome+isolation+for+the+discovery+of+kidney+disease+biomarkers.">Comparison of protein, microRNA, and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers.</a> Kidney Int. Nov. 82 (9), 1024-1032 (2012).</li><li>Kang, S. W., Kim, Y. W., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+association+of+-667+aquaporin-2+(AQP-2)+A/G+promoter+polymorphism+with+the+incidence+and+clinical+course+of+chronic+kidney+disease+in+Korea.">Study of the association of -667 aquaporin-2 (AQP-2) A/G promoter polymorphism with the incidence and clinical course of chronic kidney disease in Korea.</a> Renal Fail. 29, 693-698 (2007).</li></ol>

Bu yazı için yayın ücretleri SBI Biosciences tarafından ödendi.

Idrardan eksozom çökelmesini içeren çok yöntem Tamm-Horsfall proteini (THP) tarafından oluşturulan polimerik ağ kaldırılmasını ele alınmamıştır. Bu protein, bu varsayımsal trans ilk düşük hızlı santrifüj içinde eksozomlar idrar, büyük miktarlarda mevcuttur. Modifiye yönteminde, sonuç eksozom çökeltme öncesinde DTT kullanılarak THP azaltmıştır. Şekil 2&#39;de gösterildiği gibi, THP, daha yüksek bir miktar, proteine ​​çıkarılmasını gösteren, son yüzer taba...

İdrar eksozomlar miRNA&#39;ların 1 zenginleştirilmiş görünmektedir idrar drenaj sistemlerinin astar glomerüler Podositler, renal tübül hücreleri ve hücreler de dahil olmak üzere idrar alanı normal ve patolojik koşullar altında tüm hücre tipleri türetilen multiveziküler organları (MVB), 100 nm küçük bir iç veziküllerdir -2. Pek çok araştırmacı, sağlıklı bireylerin idrar aynı zamanda böbrek ve / veya sistematik hastalık 3-5 olan izole eksozom belirgin proteinleri tespit ettik. Eksozomlar, ultra santrifüjleme, 8-9 ya da 10-11 çökeltme ile nanomembrane filtrenin birlikte, bu ya da sukroz 6-7 olmaksızın iki aşamalı bir diferansiyel ultra-santrifüj gibi farklı yöntemler kullanılarak izole edilebilir. Biz son zamanlarda idrar eksozomlar izole ve miRNA ve protein biyomarkerları 11 algılamak için, basit, hızlı, ölçeklenebilir ve etkili bir yöntem geliştirdik. Biyomarkerler farklı biyolojik sıvıların çeşitli tespit edilebilir olsa da, urine nispeten invazif olmayan bir şekilde büyük miktarlarda elde edilebilir, çünkü, böbrek ve idrar yolu hastalıkları olan hastalar için uygun bir seçimdir. Idrar eksozomlar 6-11 izole etmek birkaç yöntem olmasına rağmen, en yaygın kullanılan prosedür% 30 sukroz yastık ile diferansiyel Ultrasantrifügasyon bağlıdır. Bu yöntem etkili değildir ve bu yönteme izole elde edilen eksozom kalitesi yüksek olsa da, teknik kendisi uzman personel gerektirir ve zaman alıcı birçok adımları ultrasantrifügasyon, zorunlu kılmaktadır. Bu, süzme ve çökeltme gibi başka yöntemler, özel bir özelliği, çökeltme reaktifinin kullanan bir de dahil olmak üzere, eksozom izolasyonu için geliştirilmiştir (örneğin, ExoQuick-TC: Sistem Biosciences, Mountain View, CA). Bu reaktif kullanılarak yağış hızlı ve nispeten kolay olmasına rağmen, izole eksozomlann saflık Ultrasantrifügasyon meth kıyasla düşüktürod. Kısa bir süre önce laboratuarımızda 11 geliştirilen ExoQuick-TC ile çökeltme yönteminin bir modifikasyonu dahil olmak üzere idrar eksozomlann izole edilmesi için altı yöntemi olarak karşılaştırılmıştır. Biz bu değiştirilmiş çöktürme yöntemi protein, miRNA ve mRNA&#39;larının ve kendisi daha hızlı ve daha Ultrasantrifügasyon uygulamak kolay oldu prosedürün daha yüksek miktarlarda vermiştir bulundu. Burada, biz kademeli bir şekilde bizim modifiye yağış yöntemi deneysel protokol açıklar ve eksozom izolasyonu diğer yöntemlerle karşılaştırıldığında bu tekniğin avantajları ve dezavantajları bir tartışma vardır. değiştirilmiş çöktürme yöntemi exosomal proteinleri ile ilişkili ve idrardan eksozom veriminin azalmasına yol açtığı bilinmektedir Tamm-Horsfall proteini ile uzaklaştırılarak elde edilmiş eksozom partiküllerinin ilk çökeltme, içerir. Biz, bu değişimler idrardan exosomal hazırlama verim ve saflıkta arttırdığı bulunmuştur.

<table border="0" cellpadding="0" cellspacing="0" width="736">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:111px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:291px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Corning 15ml Centrifuge Tube</td>
			<td style="width:111px;">Corning (Sigma Aldrich)</td>
			<td style="width:119px;">CLS430791</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Protease Inhibitor Cocktail</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:119px;">P8340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:111px;">Sorvall</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DL-dithiothreitol</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:119px;">D9779</td>
			<td>used at final concentration of 200 mg/ml</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Novex NuPAGE SDS-PAGE system (4-12 % Bis-Tris Gel 1.0 mm, 10 well</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:119px;">NP0321BOX</td>
			<td>For SDS-PAGE analysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ECL Advance Western blotting detection kit</td>
			<td style="width:111px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">RPN2135</td>
			<td>For western blot detection of biomarkers</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Exoquick-TC Reagent</td>
			<td style="width:111px;">System Biosciences (SBI)</td>
			<td style="width:119px;">EXOTC50A-1</td>
			<td>For exosome isolation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Exosome CD9 ELISA Complete Kit</td>
			<td style="width:111px;">System Biosciences (SBI)</td>
			<td style="width:119px;">EXOEL-CD9-A-1</td>
			<td>For exosome quantification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">AllPrep DNA/RNA/Protein mini kit</td>
			<td style="width:111px;">Qiagen</td>
			<td style="width:119px;">80004</td>
			<td>For DNA, RNA, Protein isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rneasy MinElute Cleanup kit</td>
			<td style="width:111px;">Qiagen</td>
			<td style="width:119px;">74204</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NanoDrop 2000 UV-Vis Spectrophotometer</td>
			<td style="width:111px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>For Protein Quantification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ProteoSilver Sliver Stain Kit</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:119px;">PROTSIL1-1KT</td>
			<td>For staining SDS-PAGE gels</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Xcell SureLock Mini-Cell Electrophoresis System</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td>For running the SDS-PAGE gels</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan microRNA assays</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td>For RT-PCR assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">qBasePlus v1.5 software</td>
			<td style="width:111px;">Biogazelle NV</td>
			<td style="width:119px;"></td>
			<td>For analysis of RT-PCR assay data</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rabbit polyclonal antibody to ALIX</td>
			<td style="width:111px;">Millipore</td>
			<td style="width:119px;"></td>
			<td>For western blot detection of biomarkers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mouse monoclonal antibody to TSG101</td>
			<td style="width:111px;">Abcam</td>
			<td style="width:119px;"></td>
			<td>For western blot detection of biomarkers</td>
		</tr>
	</tbody>
</table>

a modified precipitation method to isolate urinary exosomes

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. Urinary exosome vesicles, 40-100 nm in size, are released into the urine under normal conditions by cells from all nephron segments and may contain protein, mRNA and microRNA representative of their cell type of origin. Under conditions of renal dysfunction or injury, exosomes may contain altered proportions of these components, which may serve as biomarkers for disease. There are currently several methods available for isolation of urinary exosomes, and we have previously conducted an experimental comparison of each of these approaches, including three based on ultracentrifugation, one using a nanomembrane ultrafiltration concentrator, one using a commercial precipitation reagent and one using a modification of the precipitation technique using ExoQuick reagent that we developed in our laboratory. We found the modified precipitation method produced the highest yield of exosome particles, miRNA, and mRNA, making this approach suitable for the isolation of exosomes for subsequent RNA profiling. We conclude that the modified exosome precipitation method offers a quick, scalable, and effective alternative for the isolation of exosomes from urine. In this report, we describe our modified precipitation technique using ExoQuick reagent for isolating exosomes from human urine.

İdrar eksozomlar böbrek epitel hücrelerinden salgılanan protein MiRNA ve mRNA biyolojik işaretleri taşırlar. Bu eksozomlann izolasyon ve algılama gibi diyabetik nefropati gibi böbrek hastalıklarının erken teşhisi için yararlı bir tanısal araç sağlayabilir. Insan deneklerden alınan idrar örneklerinde eksozomlann izole edilmesi için bir kaç yöntem vardır. Biz daha önce idrar eksozom izolasyon altı yöntemleri karşılaştırıldığında ve protein, mRNA ve miRNA düzeyleri 11 Elde inc...

Watch this Scientific Journal Video about A Modified Precipitation Method to Isolate Urinary Exosomes at JoVE.com

A Modified Precipitation Method to Isolate Urinary Exosomes

This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.

Bir Modifiye Yağış Yöntemi İdrar eksozomlar yalıtmak için

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. ...

a-modified-precipitation-method-to-isolate-urinary-exosomes

Research

JoVE Journal

Medicine

17.5K Görüntüleme.  Translational Genomics Research Institute (TGen). This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.

Video: A Modified Precipitation Method to Isolate Urinary Exosomes

A Sensitive Method to Quantify Senescent Cancer Cells

Human cells do not indefinitely proliferate. Upon external and/or intrinsic cues, cells might die or enter a stable cell cycle arrest called senescence. Several cellular mechanisms, such as telomere shortening and abnormal expression of mitogenic oncogenes, have been shown to cause senescence. Senescence is not restricted to normal cells; cancer cells have also been reported to senesce.
	Chemotherapeutical drugs have been shown to induce senescence in cancer cells. However, it remains controversial whether senescence prevents or promotes tumorigenesis. As it might eventually be patient-specific, a rapid and sensitive method to assess senescence in cancer cell will soon be required.
	To this end, the standard &beta;-galactosidase assay, the currently used method, presents major drawbacks: it is time consuming and not sensitive. We propose here a flow cytometry-based assay to study senescence on live cells. This assay offers the advantage of being rapid, sensitive, and can be coupled to the immunolabeling of various cellular markers.

Whether senescence prevents or promotes tumorigenesis remains controversial. Since chemotherapeutical drugs can induce cancer cells to senesce, studying senescence is essential for proposing new therapies. However, the standard and broadly used &beta;-galactosidase assay presents major drawbacks. We propose here a rapid and sensitive flow cytometry-based assay to quantify senescence.

Senesent Kanser Hücreleri ölçmek için bir Hassas Yöntem

Human cells do not indefinitely proliferate. Upon external and/or intrinsic  cues, cells might die or enter a stable cell cycle arrest called senescence. ...

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Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

Modifiye Manşet Tekniği Sıçangiller Servikal Kalp Nakli Modeli

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

Farelerde Mesane Ağrı için bir model olarak Mesane Distansiyonunun Uyarılmış Visceromotor Cevapları

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

A Modified Method for Heterotopic Mouse Heart Transplantion

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents available. A difficulty is presented due to the small size of the animal and the considerable technical challenges of the microsurgery involved in heart transplantation. In particular, a high rate of technical failure early after transplantation may result from recipient death and post-operative complications such as hind limb paralysis or a non-beating heart. Here, the complete technique for heterotopic mouse heart transplantation is demonstrated, involving harvesting the donor heart and its subsequent implantation into a recipient mouse. The donor heart is harvested immediately following in situ perfusion with cold heparinized saline and transection of the ascending aorta and pulmonary artery. The recipient operation involves preparation of the abdominal aorta and inferior vena cava (IVC), followed by end-to-side anastomosis of the donor aorta with the recipient aorta using a single running 10-0 microsuture and a similar anastomosis of the donor pulmonary artery with the recipient IVC. Following the operation the animal is injected with 0.6 ml normal saline subcutaneously and allowed to recover on a 37&#160;&#176;C heating pad. The results from 227 mouse heart transplants are summarized with a success rate at 48 hr of 86.8%. Of the 13.2% failures within 48 hr, 5 (2.2%) experienced hind limb paralysis, 10 (4.4%) had a non-beating heart due to graft ischemic injury and/or thrombosis, while 15 (6.6%) died within 48 hr.

A technique is demonstrated for the microsurgical procedure for heterotopic transplantation of hearts in mice, including simplified methods for donor harvesting and recipient vessel anastomosis.

Heterotopik Fare Kalp NAKLİ için Modifiye Yöntem

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents ...

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.

This manuscript presents a simple, yet powerful, in vitro method for evaluating smooth muscle contractility in response to pharmacological agents or nerve stimulation. Main applications are drug screening and understanding tissue physiology, pharmacology,&#160;and pathology.

Alt Üriner Sistem Farmakoloji değerlendirin bir Yöntem olarak mesane düz kas Şerit Kasılabilme

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&amp;#246;m Technique

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

Modifiye Bergström Tekniği İnsan İskelet Kası Biyopsi Prosedürleri

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

Yetişkin sıçan böbrek üzerinden olduğu gibi Glomeruli yalıtmak için verimli bir eleme yöntemi

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Ultrasonography of the Adult Male Urinary Tract for Urinary Functional Testing

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting in a significant economic and quality of life burden. Transgenic and other mouse models have been developed to recreate various aspects of this multifactorial disease; however, methods to accurately quantitate urinary dysfunction and the effectiveness of new therapeutic options are lacking. Here, we describe a method that can be used to measure bladder volume and detrusor wall thickness, urinary velocity, void volume and void duration, and urethral diameter. This would allow for the evaluation of disease progression and treatment efficacy over time. Mice were anesthetized with isoflurane, and the bladder was visualized by ultrasound. For non-contrast imaging, a 3D image was taken of the bladder to calculate volume and evaluate shape; the bladder wall thickness was measured from this image. For contrast-enhanced imaging, a catheter was placed through the dome of the bladder using a 27-gauge needle connected to a syringe by PE50 tubing. A bolus of 0.5 mL of contrast was infused into the bladder until a urination event occurred. Urethral diameter was determined at the point of the Doppler velocity sample window during the first voiding event. Velocity was measured for each subsequent event yielding a flow rate. In conclusion, high frequency ultrasound proved to be an effective method for assessing bladder and urethral measurements during urinary function in mice. This technique may be useful in the assessment of novel therapies for BPH/LUTS in an experimental setting.

We describe the use of high frequency ultrasound with contrast imaging as a method to measure bladder volume, bladder wall thickness, urine velocity, void volume, void duration, and urethral diameter. This strategy can be used to assess voiding dysfunction and treatment efficacy in various mouse models of lower urinary tract dysfunction (LUTD).

İdrar Fonksiyonel Testi için Erişkin Erkek İdrar Yolu Ultrasonografisi

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting ...

A Modified Simple Method for Induction of Myocardial Infarction in Mice

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI remodeling and evaluation of novel therapeutics. Different methods (e.g., isoproterenol treatment, cryoinjury, coronary artery ligation, etc.) have been used to induce MI. Compared with isoproterenol treatment and cryoinjury, coronary artery ligation may better reflect the ischemic response and chronic remodeling after MI. However, traditional methods for coronary ligation in mice are technically challenging. The current study describes a simple and efficient process for induction of MI in mice with readily available materials. The mouse chest skin was cut open under stable anesthesia. The heart was immediately externalized through the intercostal space after blunt separation of the pectoralis major and pectoralis minor. The left anterior descending branch (LAD) was ligated with a 6-0 suture 3 mm from its origin. Following LAD ligation, staining with 2,3,5-Triphenyltetrazolium chloride (TTC) indicated successful induction of MI and temporal changes of post-MI scar size. Meanwhile, survival analysis results showed overt mortality within 7 days after MI, mainly due to cardiac rupture. Moreover, post-MI echocardiographic assessment demonstrated successful induction of contractile dysfunction and ventricular remodeling. Once mastered, an MI model can be established in mice within 2-3 min with readily available materials.

Under adequate anesthesia, the mouse heart was externalized through the intercostal space, and myocardial infarction was successfully induced by ligating the left anterior descending artery (LAD) using materials readily available in most laboratories.

Farelerde Miyokard Enfarktüsünün İndüksiyonu için Değiştirilmiş Basit Bir Yöntem

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI ...

A Modified Vessel-Sparing Microsurgical Vasoepididymostomy

Microsurgical vasoepididymostomy (MVE) is the main surgical treatment for epididymal obstruction. The vasal vessels are ligated during MVE. However, preserving the vasal vessels during MVE might better simulate the normal physiological structure and be meaningful for patients who have undergone varicocelectomy. Nevertheless, preserving the vasal vessels might elevate the risk of increasing the tension of anastomosis, affecting the patency rate and leading to delayed postoperative bleeding. Therefore, we developed a novel vessel-sparing MVE to make it safer. Here is a summary of the improvements to the procedure. 1) The retrograde dissociation of the vasal vessels on the proximal testicular side was adopted as the main method, and the anterograde dissociation of the vasal vessels on the distal testicular side was adopted as a supplement to dissociate the vasal vessels to be preserved. This improvement ensures the blood supply to the vas deferens that will be used for anastomosis and also provides longer vasal vessels, which reduces the tension of anastomosis. 2) By fixing the vas deferens to be anastomosed and the broken end of the vas deferens, the free vasal vessels get fixed, which resolves the problem of transmission of vas tension to the vasal vessels and reduces the risk of vasal vessel hemorrhage. 3) Dissociation of the vas deferens after opening the tunica vaginalis increases the mobilization of the vas deferens, which also makes the new procedure easier to complete. The evaluation of the outcomes of this new procedure showed that no significant postoperative complications occurred in the patients, and the patency rate was no different from that of the conventional procedure. Therefore, this new, improved procedure can be considered safe, with satisfactory postoperative results.

Here, we present a protocol to preserve the vasal vessels in microsurgical vasoepididymostomy. The surgical security is enhanced by preserving the vasal vessels using a retrograde-anterograde dissociation and fixing the vasal vessels.

Modifiye Damar Koruyucu Mikrocerrahi Vazoepididimozomi

Microsurgical vasoepididymostomy (MVE) is the main surgical treatment for epididymal obstruction. The vasal vessels are ligated during MVE. However, ...