Name: real-time imaging of leukotriene b4 mediated cell migration and blt1 interactions with &beta;-arrestin
Uploaded: 2010-12-23T15:00:00
Description: Watch this Scientific Journal Video about Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with ÃŽÂ²-arrestin at JoVE.com

Araştırma Ulusal Sağlık Enstitüleri Sağlık hibe AI-52.381, CA138623 ve Kentucky Akciğer Kanseri Araştırma Kurulu ve James Graham Brown Kanser Merkezi&#39;nden kurumsal destek tarafından desteklenmektedir.

Metodoloji Mikroskop açıklaması Eclipse TE300 Nikon Ters Mikroskop bağlı TE-FM Epi-Floresan sistemi kullanılarak yapılan canlı hücre görüntüleme deneyleri. Mikroskop ısıtma sahne ile donatılmıştır. Serin bir ek bileşenini HD dijital / B CCD (Roper Bilimsel) kamera ve LAMDA 10-2 optik filtre değiştirici (Sutter aracı şirket) mikroskop eklenir. Uyarma ve emisyon dalga boylarında filtre çarklarını kontrollü ve Lamba 10-2 filtre çarkı denetle...

<ol>
	<li>Wess, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G-protein-coupled+receptors:+molecular+mechanisms+involved+in+receptor+activation+and+selectivity+of+G-protein+recognition.">G-protein-coupled receptors: molecular mechanisms involved in receptor activation and selectivity of G-protein recognition.</a> FASEB J. 11, 346-354 (1997).</li><li>Gether, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncovering+molecular+mechanisms+involved+in+activation+of+G+protein-coupled+receptors.">Uncovering molecular mechanisms involved in activation of G protein-coupled receptors.</a> Endocr Rev. 21, 90-113 (2000).</li><li>Pierce, K. L., Premont, R. T., Lefkowitz, R. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+mediator+networks+in+cell+signaling:+update+and+impact+of+cytokines.">Lipid mediator networks in cell signaling: update and impact of cytokines.</a> Faseb J. 10, 1147-1158 (1996).</li><li>Tager, A. M., Luster, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLT1+and+BLT2:+the+leukotriene+B(4)+receptors.">BLT1 and BLT2: the leukotriene B(4) receptors.</a> Prostaglandins Leukot Essent Fatty Acids. 69, 123-134 (2003).</li><li>Toda, A., Yokomizo, T., Shimizu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukotriene+B4+receptors.">Leukotriene B4 receptors.</a> Prostaglandins Other Lipid Mediat. 68-69, 575-585 (2002).</li><li>Haribabu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+the+leukotriene+B(4)+receptor+in+mice+reveals+its+role+in+inflammation+and+platelet-activating+factor-induced+anaphylaxis.">Targeted disruption of the leukotriene B(4) receptor in mice reveals its role in inflammation and platelet-activating factor-induced anaphylaxis.</a> J Exp Med. 192, 433-438 (2000).</li><li>Subbarao, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+leukotriene+B4+receptors+in+the+development+of+atherosclerosis:+potential+mechanisms.">Role of leukotriene B4 receptors in the development of atherosclerosis: potential mechanisms.</a> Arterioscler Thromb Vasc Biol. 24, 369-375 (2004).</li><li>Jala, V. R., Haribabu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukotrienes+and+atherosclerosis:+new+roles+for+old+mediators.">Leukotrienes and atherosclerosis: new roles for old mediators.</a> Trends Immunol. 25, 315-322 (2004).</li><li>Heller, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+atherogenesis+in+BLT1-deficient+mice+reveals+a+role+for+LTB4+and+BLT1+in+smooth+muscle+cell+recruitment.">Inhibition of atherogenesis in BLT1-deficient mice reveals a role for LTB4 and BLT1 in smooth muscle cell recruitment.</a> Circulation. 112, 578-586 (2005).</li><li>Miyahara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+leukotriene+B4+receptor+1+in+allergen-induced+airway+hyperresponsiveness.">Requirement for leukotriene B4 receptor 1 in allergen-induced airway hyperresponsiveness.</a> Am J Respir Crit Care Med. 172, 161-167 (2005).</li><li>Terawaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+leukotriene+B4+receptor+1+confers+resistance+to+airway+hyperresponsiveness+and+Th2-type+immune+responses.">Absence of leukotriene B4 receptor 1 confers resistance to airway hyperresponsiveness and Th2-type immune responses.</a> J Immunol. 175, 4217-4225 (2005).</li><li>Shao, W. H., Del Prete, A., Bock, C. B., Haribabu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+leukotriene+B4+receptors+BLT1+and+BLT2:+a+critical+role+for+BLT1+in+collagen-induced+arthritis+in+mice.">Targeted disruption of leukotriene B4 receptors BLT1 and BLT2: a critical role for BLT1 in collagen-induced arthritis in mice.</a> J Immunol. 176, 6254-6261 (2006).</li><li>Kim, N. D., Chou, R. C., Seung, E., Tager, A. M., Luster, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+requirement+for+the+leukotriene+B4+receptor+BLT1+for+neutrophil+recruitment+in+inflammatory+arthritis.">A unique requirement for the leukotriene B4 receptor BLT1 for neutrophil recruitment in inflammatory arthritis.</a> J Exp Med. 203, 829-835 (2006).</li><li>Jala, V. R., Shao, W. H., Haribabu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation-independent+beta-arrestin+translocation+and+internalization+of+leukotriene+B4+receptors.">Phosphorylation-independent beta-arrestin translocation and internalization of leukotriene B4 receptors.</a> J Biol Chem. 280, 4880-4887 (2005).</li><li>Jala, V. R., Haribabu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+analysis+of+G+protein-coupled+receptor+signaling+in+live+cells.">Real-time analysis of G protein-coupled receptor signaling in live cells.</a> Methods Mol Biol. 332, 159-165 (2006).</li><li>Del Prete, A., A, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+dendritic+cell+migration+and+adaptive+immune+response+by+leukotriene+B4+receptors:+a+role+for+LTB4+in+up-regulation+of+CCR7+expression+and+function.">Regulation of dendritic cell migration and adaptive immune response by leukotriene B4 receptors: a role for LTB4 in up-regulation of CCR7 expression and function.</a> Blood. 109, 626-631 (2007).</li><li>Salogni, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activin+A+induces+dendritic+cell+migration+through+the+polarized+release+of+CXC+chemokine+ligands+12+and+14.">Activin A induces dendritic cell migration through the polarized release of CXC chemokine ligands 12 and 14.</a> Blood. 113, 5848-5856 (2009).</li><li>Boudreau, J., Koshy, S., Cummings, D., Wan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+myeloid+dendritic+cells+from+bone+marrow+precursors.">Culture of myeloid dendritic cells from bone marrow precursors.</a> J Vis Exp. , (2008).</li></ol>

Canlı hücre görüntüleme, gerçek-zamanlı olarak meydana olarak spesifik proteinlerin fonksiyonu ve etkileşimleri göstermek için güçlü bir araçtır. Bu yazıda anlatılan yöntemleri açıkça LTB 4 dendritik hücrelerin hızlı göç neden olduğunu göstermektedir. Bu yöntemler sadece LTB 4 fonksiyon çeşitli hücre tipleri yönlerini genişletin, benzer yöntemlerle diğer kemokinler çeşitli uygulanan ve farklı lökosit alt popülasyonları üzerinde kemotaktik ajanlar olarak et...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cell lines:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Rat Basophilic Leukomia Cell line (RBL-2H3) or HEK293 cells.</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>CRL-2256</td>
<td>&nbsp;</td>
<tr>
<td>Media:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Delbecco's modified Eagle&#x2019;s Medium (DMEM)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11995</td>
<td>&nbsp;</td>
<tr>
<td>Phenol red free RPMI or DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11835-030</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000-044</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine (200 mM)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin (10000 U/mL)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin, 0.05% (1X) with EDTA 4Na, liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25300</td>
<td>&nbsp;</td>
<tr>
<td>HEPES (1M)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15630</td>
<td>&nbsp;</td>
<tr>
<td>Others:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>35 mm sterile glass coverslip-bottomed Fluoro dishes (0.17 mm thick) (WillCo-dish)</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>FD35-100</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Gene Pulser Cuvette (0.4 cm electrode gap) (Bio-Rad)</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>16552088</td>
<td>&nbsp;</td>
<tr>
<td>Instruments/software:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gene Pulser II electroporater </td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>TE-FM Epi-Fluorescence system attached to Nikon Inverted Microscope Eclipse TE300</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Metamorph Software</td>
<td>&nbsp;</td>
<td>Universal Imaging</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vertical Micro-pipette puller</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micro-Forge M-900</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hadraulic Micromanipulator MO-188NE</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Coarse Manual Manipulator, MN-188NE</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>cDNA constructs:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>cDNA of G-Protein coupled receptor tagged with red fluorescence protein at C-terminus (hBLT1-RFP)</td>
<td>&nbsp;</td>
<td>Jala et al 2005</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td> cDNA of cytosolic protein tagged with GFP (&beta;-arrestin1-GFP in present study).</td>
<td>&nbsp;</td>
<td>Jala et al 2005</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

real-time imaging of leukotriene b4 mediated cell migration and blt1 interactions with &beta;-arrestin

G-protein reseptörler (GPCRs) yedi transmembran protein ailesine ait olan ve hücre içi yanıtları ekstraselüler sinyallerin transdüksiyon arabuluculuk. GPCRs kontrolü gibi kemotaksis gibi çok çeşitli biyolojik fonksiyonları, hücre içi kalsiyum salınımını, ligand bağımlı bir şekilde heterotrimeric G-proteinleri 1-2 ile gen regülasyonu . Ligand bağlanması, siklik adenozin monofosfat (cAMP), inositol trifosfat (IP3) ve diacyl gliserol (DG) gibi ikinci haberciler düzeyleri modüle heterotrimeric G-proteinleri aktivasyonu önde gelen yapı değişiklikleri bir dizi neden olur. Reseptör ligand bağlama aktivasyonu ile eş zamanlı, aynı zamanda, duyarsızlaştırma, haciz ve / veya içselleştirilmesi yoluyla sinyalizasyon reseptör azaltmak için bir dizi etkinlik başlatır. GPCRs duyarsızlaştırma süreci, G-protein reseptör kinazlar (GRKs) ve β-arrestins 3 sonraki bağlanma reseptör fosforilasyon yoluyla oluşur . β-arrestins sitozolik proteinleri ve reseptör içselleştirilmesi 4-6 kolaylaştırarak var (çoğu durumda) fosforile reseptörlerine bağlanarak GPCR aktivasyon üzerine membran yerini değiştirmek. Ve LTB 4 reseptörü 2 (BLT2; düşük afinitesi reseptör; lökotrien B 4 (LTB 4), araşidonik asit yolağı ve aracılık eylemleri GPCRs, LTB 4 reseptörü 1 (afinitesi yüksek bir reseptörün BLT1) yoluyla elde edilen bir pro-inflamatuar lipid molekülünün . ) 7-9. LTB 4-BLT1 yolağı, astım, artrit ve ateroskleroz 10-17 dahil olmak üzere birçok iltihabi hastalıklar kritik olduğu gösterilmiştir . Geçerli kağıt LTB 4-kaynaklı lökosit göçü ve BLT1 etkileşimler ile β-arrestin ve mikroskopi görüntüleme teknikleri kullanılarak 18-19 canlı hücrelerde reseptör translokasyon izlemek için geliştirilen metodolojileri açıklanmaktadır. Kemik iliği elde edilen C57BL / 6 farelerinde dendritik hücreler izole edilmiş ve daha önce 20-21 açıklandığı gibi kültüre edildi. Bu hücreler, canlı hücre görüntüleme yöntemleri LTB 4 indüklenen hücre göçü göstermek için test edildi . Insan BLT1 yeşil floresan protein (β-dizi-GFP) ile etiketlenen C-terminalindeki ve β-arrestin1 kırmızı floresan proteini (BLT1 RFP) etiketli ve Rat Basophilic Leukomia (RBL-2H3) hücre hatları her iki plazmid transfekte 18-19. Bu proteinler ve yerelleştirme arasındaki etkileşimin kinetiği canlı hücre video mikroskopi kullanılarak takip edildi. Geçerli kağıt metodolojileri, canlı hücrelerin G-protein reseptörler fonksiyonel yanıtları incelemektir mikroskobik teknikler kullandıklarını açıklarlar. Reseptör kinetiği ve sitozolik protein etkileşimleri belirlemek için floresan yoğunlukları ölçmek için de geçerli kağıt Metamorph yazılım kullanımını açıklar.

Watch this Scientific Journal Video about Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with ÃŽÂ²-arrestin at JoVE.com

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with ÃƒÅ½Ã‚Â²-arrestin

Bu yazıda, özel ligandlar lökositlerin kemotaktik yanıt belirlemek ve hücre yüzey reseptörleri ve sitozolik proteinler canlı hücre görüntüleme teknikleri kullanarak arasındaki etkileşimleri tanımlamak için metodolojiyi tanımlamaktadır.

Lökotrien B Gerçek zamanlı görüntüleme 4 Hücre aracılığı ile Göç ve β-arrestin BLT1 Etkileşimleri

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

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Immunology and Infection

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

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Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

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Miyeloid hücrelerin Dynamics Gerçek zamanlı Görüntüleme<em&gt; Süm<sup&gt; Min / +</sup</em&gt; Bağırsak Tümörleri Disk Eşodaklı Mikroskopi Spinning tarafından

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

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The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Yüksek Verimli, gerçek zamanlı, Hücre içi Antimikrobiyal Aktiviteleri Çift okuma Test ve Ökaryotik Hücre Sitotoksisite

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

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Gerçek zamanlı görüntüleme ve miktar iki aşamalı dolaşım akış sistemi kullanarak mantar biyofilm gelişimi

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The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...

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Gerçek Zamanlı Hücre Analizi Kullanarak Konak Dışında Influenza Virüsünün Hayatta Kalmasını İzleme

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are ...