Sağlık / Ulusal Kanser Enstitüsü hibe (Charles J. Dimitroff RO1CA118124) Ulusal Sağlık Enstitüleri; bir Amerikan Kanser Derneği Araştırmacı Ödülü, (Charles J. Dimitroff 06-024-01-CSM) tarafından bu proje için finansman sağlanmıştır ve Sağlık / National Institute Artrit ve Kas-iskelet ve Cilt Hastalıkları hibe Ulusal Sağlık Enstitüleri (P30 AR042689 Dr. Thomas S. Kupper, Brigham ve Kadın Hastanesi, Boston, MA).

1. Akış Odası Petriler hBMEC Hazırlama (HBMEC-60 Hücreli) Monolayer <ol><li> Kat 35 37 4 geceleme 20μg/ml fibronektin 2ml (PBS) ° C veya 3 saat x 10mm doku kültürü yemekleri ° C. </li><li> Yemeklerinden fibronektin çözüm aspire edin ve [1,5 x10 5 HBMEC-60 hücre eklemek Hepes &amp; Glutamin, Orta 199% 10 FBS,% 10 İnsan Serum, 5 adet / ml heparin, 1ng/ml rekombinant insan fibroblast büyüme faktörü,% 1 penisilin Streptomisin] çanağı ve onları&gt;% 90 izdiham büyümeye izin. (2 gün sürer) </li><li> Büyüme ortamı aspire ve E-selektin ekspresyonu up-regüle, 4-6 saat için 50ng/mL IL-1β ile taze orta ekleyin. Hücre mono tabakaları artık tahlil haddeleme için hazır. E-selektin fonksiyonunu engellemek için anti-insan E-selektin Moab (klon BBIG-E4 (5D11)) 37 ~ 1hr 20μg/ml eklenebilir ° C. nötralize </li></ol> 2. Akış Odası İnsan Hematopoetik Progenitör KG1a Hücreler hazırlanması <ol><li> İnsan hematopoetik progenitör KG1a hücreleri glutamin RPMI-1640 (1x10 6 hücre / ml) confluency büyüdü ve% 10 FBS /% 1 penisilin-streptomisin ve akış odasına tayininde kullanılmak üzere şişesi doğrudan pipetlenir. </li><li> Hücre sayısı hemasitometre ve sonra da hücre 1 x 10 6 hücre / ml 10mM Hepes (H / H Tampon) ve 2mm CaCl 2 HBSS yeniden askıya alınır kullanılarak hesaplanır. </li><li> Hücreler tayininde kullanımı kadar buz üzerinde saklanır. </li></ol> 3. Mikroskop, Paralel Plakalı Akış Odası ve Şırınga Pompa (Şekil 1) hazırlanması <img src="/files/ftp_upload/old/1009_2009_1_26_7/1009_figure1.png" alt="Şekil 1" width="600" height="409" /> Şekil 1. Paralel Plakalı Akış Odası Analizi İllüstrasyon. Etkin ve tekrarlanabilir hücre analizi için en uygun düzenleme, inverted mikroskop, Harvard Şırınga Pompa, bilgisayar / kamera ve plaka paralel akış odasına yerleştirilir. <ol><li> Şırınga Pompa inverted mikroskop ve güç, güç ve ışık kaynağı açma ve mikroskop 10X Amaç seçin. </li><li> 50ml konik tüp tutucuya yerleştirin ve H / H ve 2mm CaCl 2 ile doldurun. </li><li> 50ml konik tüp altında test tüpü sahibi Yeri 5ml plastik test tüpleri </li><li> (Şırınganın pistonu ve vücut hem de güvenli olduğundan emin olun) Şırınga Pompa şırınga 60ml yerleştirin. </li><li> Stop-horoz 60ml şırınga sonu ve 3-way stop-horoz 5 ml&#39;lik şırınga takmak için 3-yollu takın. </li><li> Giriş tüp, sağ arka sol ve vakum tüpü çıkış tüpü ile mikroskop sahnede Yeri Paralel Akış Odası cihazları (GlycoTech, Inc.) </li><li> Paralel plakalı cihazları sahneye uygun izin vakum tüp vakum ve açık hava valfi takın. Hava valfi (testine akışını odasının vakum etek hava geçirmez ise) kapatın. </li><li> Çıkış boru hattı (soldaki mikroskop), 3-way stop-horoz takın. Aparat HBMEC-60 tek tabaka plaka yerleştirmek için hazırlanmıştır. </li><li> Mikroskop merkezi yerleştirin HBMEC-60 tek tabaka levha ve yeri. </li><li> Vakum ve pozisyonu tek tabaka levha merkezinde olduğu gibi plaka üzerinde akış odasına aparatı açın. </li><li> HBMEC-60 tek tabaka levha üzerine hafifçe akışını odası aparatı alt ve akış odasına aparat aşağı emme izin. (Hava kaçan hiçbir sesler ve bu noktada kauçuk conta sıkıştırmak için odasına basınç uygulamak gerekebilir.) </li><li> Vakum yeri giriş tüp 2mm CaCl 2 ile H / H içeren 50ml konik tüp içine (sağda) kurulmuş oldu. </li><li> Açık 3-way stop-horoz 60ml yer enjektör ve pompa akmasına izin verecek şekilde &quot;pompa modu.&quot; </li><li> Giriş / çıkış hatlarını hava plaka üzerinde hücrelerin kaldırmadan kaldırmak için, sıvı aparatı ulaşmadan hemen önce mL / dakika sonra hızlı bir şekilde 0.5 &#39;e ayarlanmış, Şırınga Pompa emme tüpü doldurmak için 2mL/minute kullanabilirsiniz. Bu özenle kurmak ve plaka tek tabaka kaldırma hava kabarcıkları önlemek başbakan zorunludur. </li><li> 60ml şırınga, akış odası ve Şırınga Pompa içine sıvı hareket görülür sonra astar ve Akış odasının kullanıma hazırdır. Bu set, her hücre giriş çalıştırmak için gereken her yeni plaka için tekrarlanır. </li></ol> 4. Lökosit Rolling Olaylar NIS Elements kullanarak yakalama <ol><li> Masaüstünde açık NIS elemanları. </li><li> Basın canlı görüntü simgesi &quot;oyun&quot;. Otomatik pozlama şimdi plaka daha kolay görmek için izin verir ve odaklama için önemlidir. </li><li> Mikroskop hücre tek tabaka düzgün odaklı olduğunu emin olun. </li><li> 1 kare / saniye ve ayarlamak ışık mikroskop odaklı sonra, yeniden ayarlamak maruz kalma. Görüntü bilgisayarda görünür ve artık görülebilir düşük ışık seviyesi nedeniyle mikroskop vizör gerekir. Işık histogram sonra reklam olabilirarka plan maruz kaldırmak veya hücre netliğini artırmak için justed. </li><li> Film çekimi için ideal bir görüntü elde etmek 2X2 bin veya Canlı Bin tıklayın </li><li> Menü çubuğunda &quot;Makro&quot; tıklayın ve &quot;Limana yazmak&quot; seçeneğini seçin port &quot;COM3&quot; ve &quot;açık&quot;. (Bu liman Şırınga Pompa ile koordine etmek için açılır. </li><li> Pipet KG1a hücre süspansiyonu giriş boru hattı yakınında 5ml test tüpüne 1 ml ve daha sonra giriş boru hattı test tüpünün dibine yerleştirin. </li><li> Menü çubuğu, &quot;Yakala&quot;, &quot;time-lapse edinimi.&quot; </li><li> Yeni bir menü açılır. Protokol süren 210 saniye seçin, bu HBMEC-60 Hücreler üzerinde yuvarlanan programın uzunluğu. (Her bir aşama, pompa protokol süresi boyunca her saniye bir resim çekmek için programlanmıştır. Ayrıca, pompa başlatmak için COM3 bağlantı noktası üzerinden bir komut göndermek için ayarlanır.) Zaman atlamalı kesintiye uğrarsa, pompa durmayacak kendi başına ve sıfırlanması gerekir. Canlı görüntü dönmek için İptal tuşuna basın. </li><li> Kılavuzuna göre &quot;Program Modu&quot; Şırınga Pompa Seti. (Program modu, özel akış oranları (yani kayma stres düzeyleri) odanın içinde aktarılırdı ve HBMEC-60 hücre tek tabaka üzerinde KG1a hücre etkileşimleri dikte manuel programlama için sağlar.) HBMEC Hücreler üzerinde yuvarlanan KG1a hücre ayarları 4.2 dynes 45 s / cm 2, 0.3 dynes 60 sn / cm 2 ve kademeli artışlar her 15 sn maksimum 4.2 dynes / cm 2 </li><li> Odasında duvar kayma gerilmesi (W st) aşağıdaki denkleme göre hesaplanır: G st / ml hacimsel debisi Q μ medyanın tahmini viskozite (0,0076 P) = 6μQ / 2 b, s, kanal cm yüksekliği (conta kalınlığı) (0,03 cm), ve b cm (0.5 cm) kanal genişliği. Akış hızları, Şırınga Pompa program modunu kullanarak düzenlenmiştir. </li><li> Mikroskop ve bilgisayar artık zaman atlamalı fotoğraf çekmek için hazır. Basın bilgisayarda veri elde başlamak için &quot;run start&quot;. Program çalışırken, KG1a hücreleri içeren test tüpü (hiç hava kabarcığı olmadığından emin olun) giriş / çıkış boru tam orta tutmak için ortam eklemek için emin olun. </li></ol> 5. Terminal Sialylation ve E-selektin aracılığı ile KG1a Hücre 5 için Yüzey Glikoprotein Bağımlılığı. (5,1-5,5, Video Klipler Şekil 2) Rolling <ol><li> KG1a hücre üzerinde yuvarlanan bir Assaying olmayan uyarılmış HBMEC-60 hücreleri (Negatif kontrol) KG1a hücreleri hazırlanan ve üzerinde akış odasına infüze edildi canlı olmayan IL-1β-uyarılmış HBMEC-60, yukarıda açıklandığı gibi . </li><li> HBMEC-60 hücreleri IL-1β ile önceden tedavi üzerinde yuvarlanan KG1a hücre Assaying KG1a hücreleri (E-selektin bağımlılık kontrolü) akış odasına üzerine hazırlanan ve infüze edildi canlı IL-1β uyarılmış yukarıda açıklandığı gibi HBMEC-60. </li><li> IL-1β ve sonra nötralizan anti-insan E-selektin Moab (E-selektin bağımlılık kontrolü) KG1a hücreleri canlı üzerinden akış odasına hazırlanan ve infüze edildi IL-ön-tedavi HBMEC-60 hücreleri üzerinde yuvarlanan KG1a hücre Assaying Yukarıda açıklandığı gibi, anti-insan E-selektin Moab nötralize 1β-uyarılmış HBMEC-60 ön işlem. </li><li> IL-1β-uyarılmış HBMEC-60 haddeleme sialidase sindirilir KG1a hücre Assaying. 37 1hr için nöraminidaz 0.1U/ml Vibrio Cholerae ° C ile ön tedavi KG1a hücreleri akış odasına üzerine hazırlanan ve infüze edildi canlı IL-1β-uyarılmış yukarıda açıklandığı gibi HBMEC-60. </li><li> IL-1β-uyarılmış HBMEC-60 haddeleme proteaz sindirilir KG1a hücre Assaying. Proteaz ön işlem KG1a hücreleri, 37 1hr için 0.2U/ml Bromelain ° C, akış odasına üzerine hazırlanan ve infüze edildi canlı IL-1β ile uyarılmış, yukarıda açıklandığı gibi HBMEC-60. </li></ol> 6. NIS-Elemanları-Yakalanan KG1a KG1a Hücre Hücreler ve Grafikleme Rolling Veri Analizi <ol><li> Zaman dizisi elde edildikten sonra, verileri kaydetmek. </li><li> Menü çubuğunda &quot;ölçü&quot; sekmesine gidin ve &quot;eşik değerini tanımlamalıyız.&quot; Böylece istenilen hücrelerin kırmızı renkli barlar taşıyın. &quot;Tüm görüntüleri&quot; sonra &quot;Tamam&quot; seçeneğini seçin. Tüm zaman atlamalı sırası görünür kırmızı haddeleme hücreleri ile modifiye edilmelidir. </li><li> &quot;Tedbir&quot; ve seçmek için git &quot;kısıtlamalar.&quot; (Kullanıcı haddeleme hücreleri temsil etmemektedir eşiklenir nesneler dışlamak için izin Kısıtlamalar Bu öncelikle HBMEC-60 monolayer tarafından oluşturulan arka plan) &quot;alan&quot; ve haddeleme hücreleri için alan için giriş minimum ve maksimum değerler seçin. &quot;Dairesellik&quot; uzama &quot;için bu adımı yineleyin.&quot; </li><li> &quot;Tedbir&quot; i seçin ve &quot;kısıtlamalar kullanarak ikili oluşturmak&quot; seçeneğini seçin ve haddeleme hücreleri NIS-Elements yazılımı tanıma HBMEC-60 hücreleri, tek tabaka optimize etmek için kısıtlama değerleri ayarlamak. </li><li> &quot;Ölçmek&quot;, &quot;Alan veri&quot; seçin ve ardından, &quot;reset veri&quot; seçmek için geri dönün. Bu menüyü kapatın. </li><li> &quot;Ölçü&quot; ve se dön&quot;tarama alanı&quot; bilirsiniz. </li><li> Aç &quot;Ölçü&quot;, &quot;Alan data&quot;, &quot;sayı veya Nesneler&quot; seçin ve daha sonra &quot;tüm alanlarda&quot; seçin ve Microsoft Excel&#39;e veri ihracat. </li><li> Sonraki veri toplama için NIS-Elements cep satın hazırlamak için &quot;açık veri&quot; seçin. </li></ol> Sonuçlar: <img src="/files/ftp_upload/old/1009_2009_1_26_8/1009_figure3.png" alt="Şekil 3" width="600" height="382" /> Şekil 3. Paralel Plakalı KG1a Hücreler E-selektin-Cilt Faaliyet Akış Odası Analizi. Sialidase veya proteaz ile tedavi KG1a hücreleri HBMEC-60 hücreleri IL-1β ile önceden tedavi mono tabakaları verimliliği haddeleme için analiz edildi. Negatif kontrolü haddeleme deneyler de KG1a hücreli olmayan IL-1β-uyarılmış HBMEC -60 hücreleri veya haddeleme analiz ederek yapılmıştır IL-1β uyarılan anti-insan E-selektin Moab (5D11) nötralize ile tedavi HBMEC-60 hücre. Hücre haddeleme frekansları üç nüsha olarak yapılan ve NIS-Elements yazılımı ile analiz edildi. (* İstatistiksel anlamlılık p &lt;0,001) Kayma fizyolojik stres koşulları altında endotel hücre etkileşimleri - video tabanlı olan bu raporda, lökosit analiz etmek için nasıl bir görsel tasviri. E-selektin, lökosit tethering aracılık ve haddeleme, integrinler ve hyaluronan reseptörleri gibi reseptörleri aracılığıyla ikincil daha istikrarlı bağlayıcı faaliyetlerinin taahhüt yapışkan bir davranış için gerekli ligandlar - Özellikle, E-selektin rolünü analiz. Ters bir mikroskop, Harvard Şırınga Pompa, video kamera ve bilgisayar / hücre satın alma donanım ile kullanmak için adapte paralel-plaka akış odasına kullanarak deneyler E-selektin ligand neyin yürütülen + KG1a hücreleri bizim lökosit modeli ve hBMEC hücreleri olarak olarak (HBMEC- 60), pro-inflamatuar sitokin IL-1β ile uyarılan E-selektin + insan endotel hücre modeli (1-6) olarak kullanılmıştır. Daha önce de bildirildiği gibi, biz sağlam KG1a hücre haddeleme gözlenen IL-1β-uyarılmış HBMEC-60, E-selektin-bağımlı bir şekilde (Şekil 3) (KG1a hücre haddeleme ile karşılaştırıldığında istatistiksel olarak anlamlı fark kayma stres seviyeleri aralığında hücreleri IL-1β uyarılmış HBMEC-60 hücreleri). IL-1β-HBMEC-60 hücreleri uyarılır veya haddeleme KG1a hücre oluşan negatif kontrol, IL-1β uyarılan anti-insan E-selektin mAb ile HBEMC-60 hücreleri tedavi öncesi bağımlılığını göstermek için paralel olarak yapılmıştır yuvarlanma aktivite için sitokin uyarımı ve E-selektin ekspresyonu (Şekil 3). Vibrio cholerae ön işlem KG1a hücrelerinin Ayrıca, haddeleme analiz nöraminidaz (sialidase) veya bromelain, yüzey E-selektin glikoprotein ligand sindirerek bilinen bir non-spesifik proteaz, E-selektin ligand için terminal sialik asit artıkları ve yüzey glikoprotein önemini vurguladı aktivitesi (2,3).

<ol>
	<li>Dimitroff, C. J., Kupper, T. S., Sackstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+leukocytic+migration+to+inflamed+skin+with+a+novel+fluorosugar+modifier+of+cutaneous+lymphocyte-associated+antigen.">Prevention of leukocytic migration to inflamed skin with a novel fluorosugar modifier of cutaneous lymphocyte-associated antigen.</a> J. Clin. Invest. 112, 1008-1018 .</li><li>Dimitroff, C. J., Lechpammer, M., Long-Woodward, D., Kutok, 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=Rolling+of+human+bone-metastatic+prostate+tumor+cells+on+human+bone+marrow+endothelium+under+shear+flow+is+mediated+by+E-selectin.">Rolling of human bone-metastatic prostate tumor cells on human bone marrow endothelium under shear flow is mediated by E-selectin.</a> Cancer Res. 64, 5261-5269 (2004).</li><li>Dimitroff, C. J., Descheny, L., Trujillo, N., Kim, R., Nguyen, V., Huang, W., Pienta, K. J., Kutok, J. L., Rubin, 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+of+leukocyte+E-selectin+ligands,+P-selectin+glycoprotein+ligand-1+and+E-selectin+ligand-1,+on+human+metastatic+prostate+tumor+cells.">Identification of leukocyte E-selectin ligands, P-selectin glycoprotein ligand-1 and E-selectin ligand-1, on human metastatic prostate tumor cells.</a> Cancer Res. 65, 5750-5760 (2005).</li><li>Descheny, L., Gainers, M. E., Walcheck, B., Dimitroff, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ameliorating+Skin-Homing+Receptors+on+Malignant+T+cells+with+a+Fluorosugar+Analog+of+N-acetylglucosamine:+P-selectin+Ligand+is+a+More+Sensitive+Target+than+E-selectin+Ligand.">Ameliorating Skin-Homing Receptors on Malignant T cells with a Fluorosugar Analog of N-acetylglucosamine: P-selectin Ligand is a More Sensitive Target than E-selectin Ligand.</a> J Invest Dermatol. 126 (9), 2065-2073 (2006).</li><li>Gainers, M. E., Descheny, L., Barthel, S. B., Liu, L., Wurbel, M., Dimitroff, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin-homing+receptors+on+effector+leukocytes+are+differentially+sensitive+to+glyco-metabolic+antagonism+in+allergic+contact+dermatitis.">Skin-homing receptors on effector leukocytes are differentially sensitive to glyco-metabolic antagonism in allergic contact dermatitis.</a> J. Immunology. 179 (12), 8509-8518 (2007).</li><li>Rood, P. M., Calafat, J., von dem Borne, A. E., Gerritsen, W. R., van der Schoot, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+of+human+bone+marrow+endothelial+cells:+characterisation+of+new+cell+lines.">Immortalization of human bone marrow endothelial cells: characterisation of new cell lines.</a> Eur J Clin Invest. 30 (7), 618-629 (2000).</li></ol>

Yazarlar, faiz veya açıklamalar herhangi bir çakışma var.

Paralel plakalı akış segment analizi lökosit eğitim için kullanılan in vitro test sisteminde özel bir benzer kayma gerilmesi koşulları altında endotel yapışkanlı etkileşimleri bir post-kapiller venule yüzeyinde aktarılmamaktadır. E-selektin, lökosit tethering aracılık ve aktif mikrovasküler endotel yüzey üzerinde yuvarlanan ligandlar - burada bizim görsel sunum tasvir, E-selektin rolünü araştırmak için bu sistemin değerini göstermek. Ayrıca, proteaz, glycosidase ve bu sistem içinde E-selektin ve ligandlar rol nedenlerine açıklık için engelleme antikor tedavileri uygulamanın ortaya koymaktadır. Bu analizler son derece tekrarlanabilir ve E-selektin eğitim için deneysel bir temelini oluşturan yardımcı in vivo olarak E-selektin ligand fonksiyonu. Sitokin aktive mikrovasküler endotel hücreleri (hBMEC veya insan umbilikal ven veya dermal mikrovasküler endotel hücreleri) mono tabakaları ile hücre adezyon okuyan yanı sıra, yazı bir substrat olarak rekombinant E-selektin, insan ya da arıtılmış E-selektin-Ig kimerik molekülleri kullanılarak yapılabilir hücre adezyon. Diğer yapıştırıcı etkileşimleri aracılığıyla lökosit (P) (L)-selektin ve trombosit-selektin de paralel plakalı akış odasına teknolojisi kullanılarak test edilmelidir. Tip hücre tek tabaka / protein substrat değiştirerek ya da giriş lökosit / selektin transfectant hücre modelleri, araştırmacılar, fizyolojik kayma gerilmesi koşullar altında bu etkileşimlerin rolü eğitim görebilirsiniz.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>F6178-500ML</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14190</td>
<td>&nbsp;</td>
<tr>
<td>0.5 M EDTA</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15575-038</td>
<td>&nbsp;</td>
<tr>
<td>1M HEPES</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15630</td>
<td>&nbsp;</td>
<tr>
<td>Parallel-Plate Flow Chamber Kit</td>
<td>&nbsp;</td>
<td>GlycoTech</td>
<td>31-001</td>
<td>&nbsp;</td>
<tr>
<td>PHD2000 Programmable Syringe Pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>PHD 2000</td>
<td>&nbsp;</td>
<tr>
<td>35x10mm Tissue Culture Dish</td>
<td>&nbsp;</td>
<td>BD FALCON</td>
<td>353001</td>
<td>&nbsp;</td>
<tr>
<td>Fibronectin , from human plasma</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>86088-83-7 F2006-2MG</td>
<td>&nbsp;</td>
<tr>
<td>Interleukin-1_, Human; Recombinant</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>I9401-5UG</td>
<td>&nbsp;</td>
<tr>
<td>HBSS (Hank s Buffered Saline Solution)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14170</td>
<td>&nbsp;</td>
<tr>
<td>Medium 199 w/ HEPES & Glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12320-039</td>
<td>&nbsp;</td>
<tr>
<td>Human Serum</td>
<td>&nbsp;</td>
<td>Innovative Research, Inc.</td>
<td>IPLA-SER1</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Abraxis Pharmaceutical Products</td>
<td>504011</td>
<td>&nbsp;</td>
<tr>
<td>Recombinant human fibroblast growth factor</td>
<td>&nbsp;</td>
<td>R&D Systems</td>
<td>233-FB</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin Streptomycin</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Plastic Test tubes</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>352008</td>
<td>&nbsp;</td>
<tr>
<td>Anti-human E-selectin clone BBIG-E4(5D11)</td>
<td>&nbsp;</td>
<td>R&D Systems	</td>
<td>BBA16</td>
<td>&nbsp;</td>
<tr>
<td>Bromelain</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>B-4882</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
				 	
	

		</div>

analysis of physiologic e-selectin-mediated leukocyte rolling on microvascular endothelium

E-selektin, lökosit kutanöz, kemik ve iltihaplı dokulara dolaşan işe başlatmak yardımcı mikrovasküler endotel hücreleri üzerinde bir tip-1 membran protein. E-selektin ekspresyonu, deri ve kemik mikrodamarlar kurucu ve iltihaplı dokularda / IL-1α ve TNF-α, mikrodamarlar gibi pro-inflamatuar sitokinler, indüklenebilir. Bu lektin reseptör aracılık, karakteristik bir haddeleme davranış sonucu dolaşan lökositlerin, karbonhidrat karşı reseptör ligandlar ile zayıf bağlayıcı etkileşimleri. Çünkü bu etkileşimlerin daha istikrarlı bir yapışkan olaylar ve diapedesis aktivite, lökosit yuvarlanma faaliyet karakterizasyonu ve E-selektin, lökosit ligand belirlenmesi öncesinde, lökosit kaçakçılığı ve inflamasyon çalışmaları ve anti-inflamatuar terapötikler geliştirme (1-5) büyük hedefler var . Bu raporun amacı, E-selektin, E-selektin ligand etkileşimleri fizyolojik kan akışını koşullar altında çalışmak için en yaygın olarak kullanılan teknoloji görsel, kapsamlı bir tanım vermektir. Harvard Cilt Hastalıkları Araştırma Merkezi ile birlikte laboratuar dijital görselleştirme ve yeni kayıt yazılımı, NIS-Elements eşliğinde bir devlet-the-art paralel plaka akış odasına aparat kullanır. Bu teknoloji gerçek zamanlı olarak ekranda görselleştirme yanı sıra bir video formatında kayıt haddeleme aktivite için yapışma olayları analiz etmek için izin verir. Frekans, kayma direnci haddeleme ve bağlayıcı / tethering verimliliği gibi hücre adezyon parametreleri, bir Excel elektronik tablo ihraç ve istatistiksel analize tabi, NIS-Elements yazılımı ile hesaplanır. Burada sunulan gösteri, paralel plaka akış odası, canlı, insan kemik iliği endotel hücreleri (hBMEC) bağımlı E-selektin, lökosit yuvarlanma faaliyet araştırmak için kullandı. Ölümsüzleştirdi hBMEC hücre hattı HBMEC-60 hücreleri, endotel hücre modeli (6) olarak kullanılır iken, E-selektin ligand yüksek düzeyde bir ifade hematopoetik progenitör İnsan KG1a hücreler, bizim lökosit model olarak kullanılmıştır. Akış odasında, doğal E-selektin ekspresyonu neden ve simüle HBMEC-60 hücrelerinde IL-1 ile aktive edildi. Bizim video sunumu paralel plaka akış analizi proteaz veya glycosidase uygulamak suretiyle tespit edilebilir akış odasında fizyolojik E-selektin aracılı lökosit yuvarlanma faaliyetleri ve lökosit E-selektin ligand (lar) bu fonksiyonel karakterizasyonu eğitimi için uygun bir yöntem gösterdi sindirimler.

Watch this Scientific Journal Video about Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium at JoVE.com

Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium

Bu rapor, görsel bir tasviri lökosit leşimlerini kayma fizyolojik stres altında çalışmak için paralel plaka akış segment analizi sağlar. Bu yöntem, tetik lökosit endotel hücre yüzeyleri üzerinde yuvarlanan E-selektin, endotel (E)-selektin ve lökosit ligandlar rolünü araştırmak için özellikle yararlıdır.

Mikrovasküler Endotel Fizyolojik E-selektin aracılığı ile Lökosit Rolling Analizi

E-selectin is a type-1 membrane protein on microvascular endothelial cells that helps initiate recruitment of circulating leukocytes to cutaneous, bone ...

analysis-physiologic-e-selectin-mediated-leukocyte-rolling-on

Research

JoVE Journal

Biology

19.0K Görüntüleme.  Brigham and Women's Hospital. Bu rapor, görsel bir tasviri lökosit leşimlerini kayma fizyolojik stres altında çalışmak için paralel plaka akış segment analizi sağlar. Bu yöntem, tetik lökosit endotel hücre yüzeyleri üzerinde yuvarlanan E-selektin, endotel (E)-selektin ve lökosit ligandlar rolünü araştırmak için özellikle yararlıdır.

Video: Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Nörotoksisite Yüksek İçerik Analizi Nöronal ve Astrosit Hücre Co-Kültür Testi

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biyoloji

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

Bağışık yetmezliği olan farelere Biyomühendislik İnsan Mikrovasküler Ağları

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

RNA-seq Trombin-tedavi içinde Transcriptomes Analizi ve Kontrol İnsan Pulmoner mikrovasküler endotel hücrelerinde

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

Fare Direnç arterlerden mikrovasküler endotel Tüpler İzolasyonu

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Ekstraselüler Asit Üretim Ölçüm ve Analiz Glikolitik Hızı belirleme

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Siteye özel Protein lizin asetilasyon ve Succinylation veri bağımsız edinme kütle spektrometresi kullanılarak Stoichiometry miktar

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Kemik iliği türevi fare dendritik hücreleri ve makrofajlar floresan Fusion proteinlerin ifade

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Hücre içi kalsiyum ve taze izole ve olduğu gibi fare serebral endotel membran potansiyeli eşzamanlı ölçümler

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

In Vitro E3 Ubiquitin Ligase fonksiyonunun analizi

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.