Bartly J. Mondino MD, Jules Stein Göz Enstitüsü, UCLA Direktörü ve Ramiro Ramirez-Solis, Jacqui Beyaz ve Sanger Enstitüsü, Wellcome Trust Genom Kampüsü&#39;nde Jeanne Estabel Körlük Engellenir Araştırma. Bu araştırma Oftalmik ve Görsel Araştırma Hayvanları Kullanım ARVO Bildirimi ile uyumludur.

1. Künt diseksiyon: sabitlenmemiş örneklerinde fare göz Enükleasyon <ol><li> Maruz kalma ve posterior dünya (gözün) yüzey erişimi geliştirmek için göz kapaklarını açarak çekin. </li><li> Yörüngede (altında) dünya (göz çukuru) arkasında eğik pansuman forcep yerleştirin. Mahajan Sharptip pansuman forcep (malzeme ve reaktiflerin tabloya bakınız) bu adımı kolaylaştırmak için sivri uçlu özel bir araçtır. </li><li> Forcep kapatın ve dünya sıkma önlemek için dikkatli olmak ise dünyanın arkasında bağ dokusunda ve optik sinir kavramak. </li><li> Yavaşça yukarı forcep çekin ve yörüngeden küresi koparmak. Beyaz iplik benzeri doku optik sinir olduğunu. </li><li> PBS içinde göz yerleştirin. 30-gauge iğne kullanarak, tek bir delinme küresi içine iğne 1-2 mm takarak limbus hemen posteriorunda yara olun. Bu göz dokusuna nüfuz çekemeyen gibi glutaraldehit gibi fiksatif,, en doğrudan sağlarter göz. Bir sabitleştirici, örneğin paraformaldehit veya alkol gibi, ilgi göz dokusu içine difüzyon yeteneğine sahipse bu delme yara bölgesinin oluşturulması gerekli olmayabilir. </li><li> Hemen daldırma fiksasyonu için fiksatif içine tüm göz yerleştirin. </li></ol> 2. Keskin Diseksiyon: fare gözü aşağıdaki perfüzyon fiksasyon Enükleasyon <ol><li> Uzakta dünyanın gelen göz kapağı tutmak için kavisli bir colibri forcep kullanın. </li><li> Dünya paralel Orient kavisli Westcott diseksiyon makas, yörüngenin arkasına doğru hedeflerken. </li><li> Inferior, medial, üstün, ve lateral taraftan dünya çevreleyen sabit bağ dokusu kesin. </li><li> Dünyanın arkasındaki kavisli forcep yerleştirin, dünya üzerinde basmadan bağ dokusu tutun ve gözün enükleasyonu için ileriye doğru çekin. Orbital bağ doku perfüzyonunun tespit edildiği sert olduğu için, orbital doku ilave kesme topak tamamen serbest bırakmak için gerekli olabileceke. </li></ol> 3. Fiksasyon ve Paketleme Sabit dokuların Nakliye uygun etiket ve ambalaj dahil olmak üzere kurumsal ve posta hizmet gereksinimleri takip etmelidir. Aşağıdaki protokol, ortam sıcaklığında bir enfeksiyöz olmayan biyolojik numune nakliye bir örnektir. Bu 3 ambalaj katmanları ama hiçbir A / B sınıfı biyolojik madde veya sıvı azot etiketleri gerektirir. <ol><li> Tespit kısa olması durumunda, en az 3 mL sabitleştirici, ya da post-sabitleştirici tampon ile 5 mililitrelik bir cam sintilasyon şişesi içine göz yerleştirin. Şişenin kapağı üzerinde takip numarasını yazın ve Parafilm ile kapayın. </li><li> Biyolojik numune içine yerleştirin sintilasyon şişeleri koliyi onayladı. Örnek içine yerleştirildiği bir kapalı kap, bir emici orta tabaka ve bir dış koruyucu katman: Bu enstitü, örneğin, uygun kaplar üç tabaka gerektirir. </li><li> Bir kabarcık şal paket içine ve sonra app içine bir kap yerleştirinuygun dokümantasyon ile birlikte roved laboratuar konteyner nakliye. </li></ol> 4. Temsilcisi Sonuçlar Gözlerin Histolojik incelemede Hematoksilen-Eozin boyama, enzimatik ifade algılama, transmisyon elektron mikroskobu ve immünhistokimya gibi çeşitli yöntemler ile gerçekleştirilebilir. % 4 paraformaldehid immersiyon fiksasyon sonrasında doku parafin ve mikrotom üzerinde kesit içinde gömülü idi. Yüksek verimli fenotipleme, biz öğrenci-optik sinir bölümleri analiz, göz örnek tüm doku tipleri (Şekil 1). Doku bölümleri de lacZ ifade (Şekil 2), hücresel organel transmisyon elektron mikroskobu (Şekil 3) ve antikorlar (Şekil 4) ile spesifik moleküllerin ekspresyonu incelendi. Bizim fenotipi tarama yöntemi olarak, (Latera nazal göre (medial canthus) ve zamansal ile gözün yönünü korumak için önemli değildil canthus) taraf. Göz yönelimi gerekli ise en az iki seçenek vardır. Enükleasyon sonra, bir el koter cihazı ile üç pozisyonunda temporal korneaya ışık koter uygulamış. Lezyon histoloji açıktır ve histolojik mürekkepler gibi işleme sırasında kaybolmak yok. Downside koter zararlardan muayene için kritik bir doku olabilir kornea olduğunu. Diğer seçenek ekstraoküler kas eklemeleri belirleyebilir yetenekli bir anatomist gerektirir. Stereomikroskopta altında, inferior ve superior oblik kas ekleme belirlenmesi dünyanın inferior ve superior ve yönü işaretler. 3. Her iki yönelim yöntemi ile, bir gözün sağ veya sol olarak numune izlemek için önemlidir. Birlikte, bu kesit öncesinde tam küre yönelim sağlayacaktır. <img src="/files/ftp_upload/3184/3184fig1.jpg" alt="Şekil 1" /> Şekil 1. Fare göz histolojik görüntüler afte<html> r uzaktan edinimi. Fare gözler% 4 paraformaldehit ile perfüzyon fiksasyon izleyerek keskin diseksiyon ile iki parçaya ayrıldı. Hematoksilen ve eosin ile boyandı A. Öğrenci-optik sinir bölümünde tüm doku altyapılar korunması gösterir. Optik sinir, ON. ; IPL, iç pleksiform tabaka; INL, iç nükleer tabakada; OPL, dış pleksiform tabaka; onl, dış RGC, retina ganglion hücrelerinin: Normal retina (yeşil ölçek çubuğu = 500 mikron) B. Daha yüksek bir büyütme ile laminar yapısını gösterir nükleer tabaka (fotoreseptör hücre) (ok); OS, fotoreseptör dış segmentleri;; RPE, retina pigment epiteli, C, koroid, fotoreseptör iç segment (ok başı) IS. (Yeşil ölçek bar = 50 mikron) C. Normal retina ile karşılaştırıldığında, bu numune fotoreseptör dejenerasyonu için tiner kaynaklanmaktadır. Onl, IS ve OS (ok) tamamen bulunmaz. (Yeşil ölçek bar = 50 mikron) &quot;Alt =&quot; Şekil 2 &quot;/&gt; Transgenik farelerde Şekil 2. LacZ ekspresyonu. Gözler (5 mM K3Fe (CN) 6, 5 mM K4Fe (CN) 6, 2 mM MgCl2,% 0.02 NP40 diseksiyon ile çıkarıldı ve 1 mg / ml X-gal çözelti içerisinde bekletilmiştir , 37 geceleme ve% 0,1 sodyum deoksikolatın) ° C Gözler 30 dakika ve bir mikrotom üzerinde kesit için% 2 formaldehyde/0.2% gluteraldehit ile tespit edildi. X-gal ürün (mavi) etiketler: A. Silier epiteli, B. kornea epitel ve C retina retina ganglion hücre (RGC) katmanı. INL, iç nükleer tabakada; onl, dış nükleer tabaka. <img src="/files/ftp_upload/3184/3184fig3.jpg" alt="Şekil 3" /> Şekil 3,. Retina pigment epiteli (RPE) Transmisyon elektron mikroskobu. Bir delinme yara künt diseksiyon ve yaratıldıktan sonra, gözleri daldırma vardı 0.1M sodyum fosfat tamponlu% 2.5 lik Paraformaldehyde /% 2.5 glutaraldehit ile tespit edildi. Gözler secon edildidarily,% 1 osmiyum textroxide sabit giderek susuz ve Spurr en reçine gömülü. Doku 90 nm&#39;de kesitli ve Formvar kaplı bakır yuvası ızgaralar üzerine yerleştirilir ve bir transmisyon elektron mikroskobu ile görüntülendi. Mikrografı, fotoreseptör dış nörosensöriyel retinanın kesimleri, retina pigment epiteli içindeki melanozom (M) ve Bruch membranı gösterir. <img src="/files/ftp_upload/3184/3184fig4.jpg" alt="Şekil 4," /> Şekil 4. Fare vitreusta süperoksit dismutaz-3 immünhistokimyasal etiketleme (yeşil). Gözler künt diseksiyon ile çıkarıldı ve% 4 paraformaldehid daldırma fiksasyon uygulandı. Onlar mikrotom kullanılarak parafin ve kesitli gömüldü. 01:50 seyreltilmiş Doku kesitleri tavşan poliklonal SOD3 antiserum ile inkübe edildi. SOD3 ifade keçi anti-tavşan IgG (H + L)-Alexa Fluor 488 konjuge sekonder antikor ile tespit edildi. SOD3 Etiketlenmesi gre gösterilirtr.

<ol>
	<li>Song, B. J., Tsang, S. H., Lin, C. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+models+of+retinal+degeneration+and+targets+for+gene+therapy.">Genetic models of retinal degeneration and targets for gene therapy.</a> Gene Ther. Mol. Biol. 11, 229-262 (2007).</li><li>Mahajan, V. B., Mondino, B. J., Tsang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+Mouse+Eye+Phenomics+System.">A high-throughput Mouse Eye Phenomics System.</a> Cold Spring Harbor Laboratories, Mouse Genetics Meeting. , (2010).</li><li>Smith, R. S., John, S. W. M., Nishina, P. M., Sundberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In Research Methods For Mutant Mice. , (2002).</li><li>Chang, B., Hawes, N. L., Hurd, R. E., Davisson, M. T., Nusinowitz, S., Heckenlively, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+degeneration+mutants+in+the+mouse.">Retinal degeneration mutants in the mouse.</a> Vision Res. 42, 517-525 (2002).</li><li>Anderson, M. G., Smith, R. S., Hawes, N. L., Zabaleta, A., Chang, B., Wiggs, J. L., John, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+genes+encoding+melanosomal+proteins+cause+pigmentary+glaucoma+in+DBA/2J+mice.">Mutations in genes encoding melanosomal proteins cause pigmentary glaucoma in DBA/2J mice.</a> Nat. Genet. 30, 81-85 (2002).</li><li>Hawes, N. L., Smith, R. S., Chang, B., Davisson, M., Heckenlively, J. R., John, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+fundus+photography+and+angiography:+a+catalogue+of+normal+and+mutant+phenotypes.">Mouse fundus photography and angiography: a catalogue of normal and mutant phenotypes.</a> Mol. Vis. 5, 22-22 (1999).</li><li>Won, J., Shi, L. Y., Hicks, W., Wang, J., Hurd, R., Naggert, J. K., Chang, B., Nishina, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+resources+for+vision+research.">Mouse model resources for vision research.</a> J. Ophthalmol. 2011, 391384-391384 (2011).</li><li>Pinto, L. H., Vitaterna, M. H., Siepka, S. M., Shimomura, K., Lumayag, S., Baker, M., Fenner, D., Mullins, R. F., Sheffield, V. C., Stone, E. M., Heffron, E., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Results+from+screening+over+9000+mutation-bearing+mice+for+defects+in+the+electroretinogram+and+appearance+of+the+fundus.">Results from screening over 9000 mutation-bearing mice for defects in the electroretinogram and appearance of the fundus.</a> Vision. Res. 44, 3335-3345 (2004).</li><li>Heckenlively, J. R., Winston, J. V., Roderick, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+mouse+retinal+degenerations.+I.+Correlation+of+indirect+ophthalmoscopy,+electroretinograms,+and+histology.">Screening for mouse retinal degenerations. I. Correlation of indirect ophthalmoscopy, electroretinograms, and histology.</a> Doc. Ophthalmol. 71, 229-239 (1989).</li><li>Tsang, S. H., Gouras, P., Yamashita, C. K., Kjeldbye, H., Fisher, J., Farber, D. B., Goff, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+degeneration+in+mice+lacking+the+gamma+subunit+of+the+rod+cGMP+phosphodiesterase.">Retinal degeneration in mice lacking the gamma subunit of the rod cGMP phosphodiesterase.</a> Science. 272, 1026-1029 (1996).</li><li>Tsang, S. H., Woodruff, M. L., Jun, L., Mahajan, V., Yamashita, C. K., Pedersen, R., Lin, C. S., Goff, S. P., Rosenberg, T., Larsen, M., Farber, D. B., Nusinowitz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+carrying+the+H258N+mutation+in+the+gene+encoding+the+beta-subunit+of+phosphodiesterase-6+(PDE6B)+provide+a+model+for+human+congenital+stationary+night+blindness.">Transgenic mice carrying the H258N mutation in the gene encoding the beta-subunit of phosphodiesterase-6 (PDE6B) provide a model for human congenital stationary night blindness.</a> Hum. Mutat. 28, 243-254 (2007).</li></ol>

Çıkar çatışması ilan etti.

Çoğu transgenik fareler gözleri incelemek yok laboratuvarlarda bulunması. Bizim video tekniği gözleri ile az deneyime sahip laboratuvarlar doku edinimi optimize etmek için uzaktan cerrahi beceri transferi için basit ve standartlaştırılmış bir yöntem gösterilmektedir. Bu video tekniği anlamlı karşılaştırmalı fenotipik ve moleküler çalışmalar önlemek standart olmayan doku toplama ve fiksasyon yöntemleri nedeniyle uzman sitelere sınırlı sayıda kullanımı yüksek hacimli fenotiplendirme, bü...

<table border="1"><tbody><tr><th> Isim </th><th> Şirket </th><th> Katalog Numarası </th><th> Yorumlar </th></tr><tr><td> Kavisli Giyinme Forcep </td><td> Storz Ophtalmics&#39;in </td><td> E1408 </td><td></td></tr><tr><td> Mahajan Sharptip soyunma forcep </td><td> Storz Ophtalmics&#39;in </td><td> E1406 (REF SP7-64520) </td><td></td></tr><tr><td> Kavisli Westcott Makas </td><td> Storz Ophtalmics&#39;in </td><td> E3321 WH </td><td></td></tr><tr><td> 15 ° BD Beaver Mikrocerrahi Blade </td><td> Becton-Dickinson </td><td> 374881 </td><td></td></tr><tr><td> 0.22 İnce Castroviejo dikişin Forseps </td><td> Storz Ophtalmics&#39;in </td><td> E1805 </td><td></td></tr><tr><td> 0.12 Colibri forseps </td><td> Storz Ophtalmics&#39;in </td><td> 2/132 </td><td></td></tr><tr><td> 30-gauge iğne </td><td> Becton-Dickinson </td><td> 305128 </td><td></td></tr><tr><td> Biohazard Mailer </td><td> Balıkçı </td><td> 03-523-4 </td><td></td></tr><tr><td> Parafilm </td><td> Balıkçı </td><td> 13-374-10 </td><td></td></tr><tr><td> Cam sintilasyon şişeleri </td><td> Wheaton </td><td> 4500413033 </td><td></td></tr><tr><td> PBS, pH 7.4 </td><td> Invitrogen </td><td> 70011-044 </td><td></td></tr><tr><td> % 16 paraformaldehit </td><td> Elektron Mikroskopi Bilimleri </td><td> 15700 </td><td></td></tr><tr><td> 0.1M sodyum fosfat tamponlu% 2.5 lik Paraformaldehyde /% 2.5 glutaraldehit </td><td> Elektron Mikroskopi Bilimleri </td><td> 15700 &amp; 16300 </td><td> Laboratuvarda Karışık </td></tr><tr><td> 50% Glutaraldehit </td><td> Elektron Mikroskopi Bilimleri </td><td> 16300 </td><td></td></tr><tr><td> 0.25% Formvar </td><td> Elektron Mikroskopi Bilimleri </td><td> 15810 </td><td></td></tr><tr><td> PolisSlot Izgara başına </td><td> Elektron Mikroskopi Bilimleri </td><td> M2010-CR </td><td></td></tr><tr><td> % 4 osmiyum tetroksit </td><td> Elektron Mikroskopi Bilimleri </td><td> 19140 </td><td></td></tr><tr><td> Anti-SOD3 antikor </td><td> Abcam </td><td> Ab21974 </td><td></td></tr><tr><td> Keçi anti-tavşan Alexa Fluor 488 </td><td> Invitrogen </td><td> A11070 </td><td></td></tr><tr><td> Spurr sitesi gömme reçine </td><td> Elektron Mikroskopi Bilimleri </td><td> 14300 </td><td></td></tr></tbody></table>

mouse eye enucleation for remote high-throughput phenotyping

Fare insan gözü oftalmik hastalık translasyonel çalışma için önemli bir genetik modelidir. Bu tür makula dejenerasyonu, fotoreseptör dejenerasyonu, katarakt, glokom, retinoblastom ve diyabetik retinopati gibi, insanlarda körleştiriyor hastalıklar 1-5 En transgenik ve nakavt fareler olmayan oftalmik hastalıklar çalışma laboratuarları tarafından üretilen oylandı. Transgenik farelerin değinmeyecek ama oylandı organ sistemleri arasındaki genetik koruma, aynı genlerin çoğunun da oküler geliştirme ve hastalık bir rol oynadığını düşündürmektedir. Dolayısıyla, bu farelerin gözünde yeni genotip-fenotip ilişkisi keşfetmek için önemli bir kaynak oluşturmaktadır. Bu farelerin dünyaya yayılmış olduğundan, bu satın korumak ve verimli, maliyet-etkin şekilde fenotip bunları zordur. Böylece, en yüksek verim oftalmik fenotipleme ekranlarında canlı farelerde gözler incelemek, oftalmik uzmanlığı bünyesinde gerektiren birkaç yerle sınırlıdır. 6-9 laboratuar tarafından geliştirilen diğer bir yaklaşım, transgenik fare gözünde büyük ya da küçük-çaplı araştırmalar kullanılabilir uzak doku-kazanılması için bir yöntemdir. Video tabanlı cerrahi beceri transferi, doku fiksasyonu ve nakliye için Standartlaştırılmış prosedürler herhangi bir laboratuar mutant hayvanlar kadar tüm gözler toplamak ve moleküler ve morfolojik fenotipleme için bunları göndermek için izin verir. Bu video makalede, uzaktan fenotipleme analizleri için sabitlenmemiş ve perfüzyon hem sabit fare gözler tanıttılar ve aktarmak için teknikler mevcut.

Watch this Scientific Journal Video about Mouse Eye Enucleation for Remote High-throughput Phenotyping at JoVE.com

Mouse Eye Enucleation for Remote High-throughput Phenotyping

Diseksiyon tekniği high-throughput ekranlarında fenotipleme gerçekleştirmek için doku fiksasyonu fare gözün enükleasyonu göstermektedir.

Uzaktan Yüksek throughput fenotipleme için Fare Göz Enükleasyon

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

mouse-eye-enucleation-for-remote-high-throughput-phenotyping

Research

JoVE Journal

Medicine

20.2K Görüntüleme.  University of Iowa. Diseksiyon tekniği high-throughput ekranlarında fenotipleme gerçekleştirmek için doku fiksasyonu fare gözün enükleasyonu göstermektedir.

Video: Mouse Eye Enucleation for Remote High-throughput Phenotyping

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive, low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics.
The combination of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation, production host cells are first transfected with an expression vector carrying the gene of interest 1, followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis 2-4. Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed, these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup.
This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes, the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure, this automated platform demonstrated advantages of significantly increased capacity, ensured clonality, traceability in cell line history with electronic documentation and much reduced opportunity in operator error.

A high-throughput, automated platform of manufacturing cell line development for producing protein therapeutics is described. Implementation of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has demonstrated significantly increased processing capacity in cell line development with improved cell line quality and high reproducibility.

Protein Therapeutics İmalat Hücre Hatları Kalkınma Yüksek throughput Otomatik Platformu

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing ...

Tıp

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

Anjiyogenez Çalışması Kornea Cep Testi Fare Modeli

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

Bilinçli Mesnetlenmemiş Fare hiperinsülinemik öglisemik Kelepçeleri

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

Remote Magnetic Navigation for Accurate, Real-time Catheter Positioning and Ablation in Cardiac Electrophysiology Procedures

New remote navigation systems have been developed to improve current limitations of conventional manually guided catheter ablation in complex cardiac substrates such as left atrial flutter. This protocol describes all the clinical and invasive interventional steps performed during a human electrophysiological study and ablation to assess the accuracy, safety and real-time navigation of the Catheter Guidance, Control and Imaging (CGCI) system. Patients who underwent ablation of a right or left atrium flutter substrate were included. Specifically, data from three left atrial flutter and two counterclockwise right atrial flutter procedures are shown in this report. One representative left atrial flutter procedure is shown in the movie. This system is based on eight coil-core electromagnets, which generate a dynamic magnetic field focused on the heart. Remote navigation by rapid changes (msec) in the magnetic field magnitude and a very flexible magnetized catheter allow real-time closed-loop integration and accurate, stable positioning and ablation of the arrhythmogenic substrate.

This report provides a detailed description of a new remote navigation system based on magnetic driven forces, which has been recently introduced as a new robotic tool for human cardiac electrophysiology procedures.

Kardiyak Elektrofizyoloji İşlemleri Doğru, gerçek zamanlı Kateter Konumlandırma ve Ablasyon için uzaktan Manyetik Navigasyon

New remote navigation systems have been developed to improve current limitations of conventional manually guided catheter ablation in complex cardiac ...

High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT).
Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2R&alpha;5, TNFR16, HGF7, IL-88, elafin2, and REG3&alpha;3 (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage. For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials.
This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents.

High throughput validation of multiple candidate biomarkers can be performed by sequential ELISA in order to minimize freeze/thaw cycles and use of precious plasma samples. Here, we demonstrate how to sequentially perform ELISAs for six different validated plasma biomarkers1-3 of graft-versus-host disease (GVHD)4 on the same plasma sample.

Akut Graft-Versus-Host Hastalığı Biyomarkörler onaylanması için Yüksek Verimli Sıralı ELISA

Unbiased discovery proteomics strategies have  the potential to identify large numbers of novel biomarkers that can improve  diagnostic and prognostic ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

Nöroprotektif Stratejileri Tedavi Faktörleri teslimi için tasarlandı Erişkin Kök Hücre Yüksek Verimli Karakterizasyonu

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

Fare Retinal Sirkülasyonunda Akış Dinamikleri Çalışmak İçin Eritrositler ve Lökositlerin Floresan Boya Etiketlenmesi

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate (HoLEP).

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the quality of life. BPH includes 2 major treatments: medication and surgery. Surgical treatment is often the most effective and final intervention. Holmium laser enucleation of the prostate (HoLEP), one of the most efficient surgical procedures for BPH, is conducted transurethrally. During surgery, the most important and most difficult part is to locate the surgical capsule of the prostate, which is consistent to the idea of anatomic surgery. Different skills may be needed in treating different parts of the prostate. HoLEP tends to be efficient and safe with good hemostatic properties, and able to treat bladder calculus, which may be the complications of BPH. The technique is particularly suitable for prostate of varying volumes and sizes. Indeed, HoLEP also presents certain disadvantages, such as a long learning curve and costly equipment. Regardless, this method may be the "new standard" and the excellent method for the surgical treatment of BPH.

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate HoLEP.

Here we present a safe and efficient protocol, holmium laser enucleation of the prostate, to treat benign prostatic hyperplasia.

Benign Prostatik Hiperplazi için cerrahi tedavi: holmiyum lazer enükleasyon prostat (HoLEP).

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the ...

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

Delici cisim kaynaklı Iris neovaskülarizasyon Rubeosis Iridis bir fare modeli olarak

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.