Name: analysis of physiologic e-selectin-mediated leukocyte rolling on microvascular endothelium
Uploaded: 2009-02-11T00:00:00
Duration: 14 min 16 s
Description: Watch this Scientific Journal Video about Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium at JoVE.com

<p class="jove_content"> एक अमेरिकन कैंसर सोसायटी रिसर्च स्कॉलर अवार्ड (चार्ल्स जे Dimitroff 06-024-01-CSM) द्वारा इस परियोजना के लिए अनुदान प्रदान किया गया, स्वास्थ्य / राष्ट्रीय कैंसर संस्थान अनुदान (RO1CA118124 चार्ल्स जे Dimitroff) के राष्ट्रीय संस्थानों, और स्वास्थ्य / राष्ट्रीय गठिया और Musculoskeletal संस्थान और त्वचा रोग अनुदान के राष्ट्रीय संस्थान (p30 डॉ. थॉमस एस Kupper AR042689, ब्रिघम और महिला अस्पताल, बोस्टन, MA). </p>

<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> <em style='font-style: italic'>J. Clin. Invest</em>. <b>112</b>, 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> <em style='font-style: italic'>Cancer Res</em>. <b>64</b>, 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> <em style='font-style: italic'>Cancer Res</em>. <b>65</b>, 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> <em style='font-style: italic'>J Invest Dermatol</em>. <b>126</b> (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> <em style='font-style: italic'>J. Immunology</em>. <b>179</b> (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> <em style='font-style: italic'>Eur J Clin Invest</em>. <b>30</b> (7), 618-629 (2000).</li></ol>

लेखकों ब्याज या खुलासे की कोई संघर्ष है.

<p class="jove_content"> समानांतर प्लेट प्रवाह चैम्बर विश्लेषण इन विट्रो परख प्रणाली में एक विशेष ल्युकोसैट अध्ययन के लिए इस्तेमाल किया है - कतरनी तनाव समान शर्तों के तहत endothelial चिपकने वाला बातचीत के बाद केशिका venule की सतह पर दिया. ल्युकोसैट tethering mediating और सक्रिय microvascular endothelium की सतह पर रोलिंग में ई selectin ligands - जैसा कि हमारे दृश्य प्रस्तुति में यहाँ दर्शाया गया है, हम ई selectin की भूमिका की जांच के लिए इस प्रणाली का मूल्य प्रदर्शित. हम यह भी protease glycosidase, और इस प्रणाली में ई selectin और अपने ligands की भूमिका elucidating के लिए अवरुद्ध एंटीबॉडी उपचार के आवेदन प्रकट करते हैं. ये विश्लेषण अत्यधिक प्रतिलिपि प्रस्तुत करने योग्य हैं और ई selectin के अध्ययन के लिए एक प्रायोगिक आधार के रूप में मदद - ई - selectin vivo में ligand समारोह. </p><p class="jove_content"> साइटोकाइन सक्रिय microvascular endothelial कोशिकाओं (hBMEC या मानव नाल की शिरा या त्वचीय microvascular endothelial कोशिकाओं) के monolayers साथ कोशिका आसंजन का अध्ययन करने के अलावा, assays पुनः संयोजक मानव ई selectin या एक सब्सट्रेट के रूप में ई selectin पुलिस महानिरीक्षक chimeric अणुओं शुद्ध का उपयोग किया जा सकता है है कोशिका आसंजन के लिए. अन्य चिपकने वाला बातचीत ल्युकोसैट (एल) selectin और प्लेटलेट (पी) selectin भी समानांतर प्लेट प्रवाह चैम्बर प्रौद्योगिकी का उपयोग कर assayed किया जा सकता है के माध्यम से मध्यस्थता. सेल सब्सट्रेट / monolayer प्रोटीन के प्रकार से अलग है या इनपुट ल्युकोसैट / selectin transfectant सेल मॉडल की, जांचकर्ताओं को शारीरिक कतरनी तनाव की शर्तों के तहत इन मुलाकातों की भूमिका का अध्ययन कर सकते हैं. </p>

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>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-selectin is a type-1 membrane protein on microvascular endothelial cells that helps initiate recruitment of circulating leukocytes to cutaneous, bone and inflamed tissues.  E-selectin expression is constitutive on dermal and bone microvessels and is inducible by pro-inflammatory cytokines, such as IL-1&#945;/  and TNF-&#945;, on microvessels in inflamed tissues.  This lectin receptor mediates weak binding interactions with carbohydrate counter-receptor ligands on circulating leukocytes, which results in a characteristic  rolling  behavior.  Because these interactions precede more stable adhesive events and diapedesis activity, characterization of leukocyte rolling activity and identification of leukocyte E-selectin ligands have been major goals in studies of leukocyte trafficking and inflammation and in the development of anti-inflammatory therapeutics (1-5).  The intent of this report is to provide a visual, comprehensive description of the most widely-used technology for studying E-selectin   E-selectin ligand interactions under physiologic blood flow conditions.  Our laboratory in conjunction with the Harvard Skin Disease Research Center uses a state-of-the-art parallel-plate flow chamber apparatus accompanied by digital visualization and new recording software, NIS-Elements.  This technology allows us to analyze adhesion events in real time for onscreen visualization as well as record rolling activity in a video format.  Cell adhesion parameters, such as rolling frequency, shear resistance and binding/tethering efficiency, are calculated with NIS-Elements software, exported to an Excel spreadsheet and subjected to statistical analysis.  In the demonstration presented here, we employed the parallel-plate flow chamber to investigate E-selectin-dependent leukocyte rolling activity on live human bone marrow endothelial cells (hBMEC).  Human hematopoietic progenitor KG1a cells, which express a high level of E-selectin ligand, were used as our leukocyte model, while an immortalized hBMEC cell line, HBMEC-60 cells, was used as our endothelial cell model (6).  To induce and simulate native E-selectin expression in the flow chamber, HBMEC-60 cells were first activated with IL-1 .  Our video presentation showed that parallel-plate flow analysis is a suitable method for studying physiologic E-selectin-mediated leukocyte rolling activities and that functional characterization of leukocyte E-selectin ligand(s) in the flow chamber can be ascertained by implementing protease or glycosidase digestions.

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

<p class="jove_content">इस रिपोर्ट में शारीरिक कतरनी तनाव के तहत ल्युकोसैट endothelial बातचीत के अध्ययन के लिए एक समानांतर थाली प्रवाह चैम्बर विश्लेषण के दृश्य चित्रण प्रदान करता है. इस विधि endothelial (ई) selectin और ल्युकोसैट ई selectin ligands कि ट्रिगर ल्युकोसैट endothelial सेल सतहों पर रोलिंग की भूमिका की जांच के लिए विशेष रूप से उपयोगी है.</p>

शारीरिक ल्युकोसैट ई selectin की मध्यस्थता रोलिंग की Microvascular endothelium पर विश्लेषण

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

Research

JoVE Journal

Biology

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

Neurotoxicity के उच्च सामग्री विश्लेषण के लिए एक neuronal और astrocyte सह संस्कृति परख

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

Bioengineering Human Microvascular Networks in Immunodeficient Mice

बायोइन्जिनियरिंग immunodeficient चूहों में मानव Microvascular नेटवर्क

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

थ्रोम्बिन इलाज में आरएनए - seq Transcriptomes का विश्लेषण और नियंत्रण मानव फेफड़े Microvascular endothelial कोशिकाओं

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

माउस प्रतिरोध धमनियों से microvascular endothelial ट्यूब का अलगाव

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

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

स्कैनिंग इलेक्ट्रॉन माइक्रोस्कोपी द्वारा जिगर Sinusoidal endothelial कोशिकाओं का विश्लेषण और उनकी Fenestrations के लिए एक मानकीकृत विधि

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

कोशिकी एसिड के उत्पादन के मापन और विश्लेषण ग्लाइकोलाइटिक दर निर्धारित करने के लिए

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

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Immunohistochemical और Immunofluorescent विश्लेषण के लिए आंतों के ऊतकों तैयारी के लिए स्विस रोलिंग तकनीक में सुधार

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

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

ठहराव of Site-विशिष्ट प्रोटीन Lysine Acetylation और Succinylation Stoichiometry का उपयोग डाटा-स्वतंत्र अधिग्रहण जन स्पेक्ट्रोमेट्री

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

Murine अस्थि मज्जा में फ्लोरोसेंट फ्यूजन प्रोटीन की अभिव्यक्ति-व्युत्पंन वृक्ष कोशिकाओं और मैक्रोफेज

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

हौसले से अलग और बरकरार माउस सेरेब्रल Endothelium में Intracellular कैल्शियम और झिल्ली क्षमता का एक साथ माप

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