Name: isolation of human umbilical vein endothelial cells and their use in the study of neutrophil transmigration under flow conditions
Uploaded: 2012-08-08T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions at JoVE.com

Vi takker Dr. Pina Colarusso og Live Cell Imaging facilitet for deres hjælp med billedbehandling og image analyse; Ms Lailey for hende teknisk bistand og enhed 51 ved foden Hospital i Calgary, AB for at give mennesker navlestrenge. Dr. KD Patel er en Alberta innoverer: Health Solutions Scientist. Dette arbejde blev støttet af et driftstilskud fra canadiske Institutes for Health Research og udstyr og infrastruktur tilskud fra den canadiske Institut for Innovation og Alberta videnskab og forskning Authority.

1. Isolering og vedligeholdelse af humane vaskulære endotelceller <ol><li> Niveau 2 biosikkerhed procedurer skal anvendes når man arbejder med humant blod og væv. Brug saks klippe ledningen fra moderkagen og derefter nøje undersøge ledningen for blodpropper og skader forårsaget af klemme i ledningen under fødslen. Klip ud og kassere disse dele af ledningen. </li><li> Fremstil frisk collagenaseopløsning ved 1 mg / ml ved at hælde 50 ml varm ledningen buffer til et rør, der indeholder 50 mg collagenase. </li><...

<ol>
	<li>Nathan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+and+immunity:+challenges+and+opportunities.">Neutrophils and immunity: challenges and opportunities.</a> Nat. Rev. Immunol. 6, 173-182 (2006).</li><li>Diacovo, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+rolling,+arrest,+and+transmigration+across+activated,+surface-adherent+platelets+via+sequential+action+of+P-selectin+and+the+beta+2-integrin+CD11b/CD18.">Neutrophil rolling, arrest, and transmigration across activated, surface-adherent platelets via sequential action of P-selectin and the beta 2-integrin CD11b/CD18.</a> Blood. 88, 146-157 (1996).</li><li>Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+site+of+inflammation:+the+leukocyte+adhesion+cascade+updated.">Getting to the site of inflammation: the leukocyte adhesion cascade updated.</a> Nat. Rev. Immunol. 7, 678-689 (2007).</li><li>Petri, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+LSP1+is+involved+in+endothelial+dome+formation,+minimizing+vascular+permeability+changes+during+neutrophil+transmigration+in+vivo.">Endothelial LSP1 is involved in endothelial dome formation, minimizing vascular permeability changes during neutrophil transmigration in vivo.</a> Blood. 117, 942-952 (2011).</li><li>Chavakis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+junctional+adhesion+molecule-C+promotes+neutrophil+transendothelial+migration+in+vitro+and+in+vivo.">The junctional adhesion molecule-C promotes neutrophil transendothelial migration in vitro and in vivo.</a> J. Biol. Chem. 279, 55602-55608 (2004).</li><li>Oh, H., Siano, B., Diamond, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+Isolation+Protocol.">Neutrophil Isolation Protocol.</a> J. Vis. Exp. (17), e745 (2008).</li><li>Liu, Y. <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+leukocyte+transmigration:+cell+surface+interactions+and+signaling+events.">Regulation of leukocyte transmigration: cell surface interactions and signaling events.</a> J. Immunol. 172, 7-13 (2004).</li><li>Alcaide, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+recruitment+under+shear+flow:+it's+all+about+endothelial+cell+rings+and+gaps.">Neutrophil recruitment under shear flow: it's all about endothelial cell rings and gaps.</a> Microcirculation. 16, 43-57 (2009).</li><li>Jutila, 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=Measurement+of+neutrophil+adhesion+under+conditions+mimicking+blood+flow.">Measurement of neutrophil adhesion under conditions mimicking blood flow.</a> Neutrophil Methods and Protocols. 412, 239-256 (2007).</li><li>Cuvelier, S. L., Patel, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+leukocyte+rolling+and+adhesion+in+vitro+under+flow+conditions.">Studying leukocyte rolling and adhesion in vitro under flow conditions.</a> Basic Cell Culture Protocols. 290, 331-342 (2005).</li><li>Wiese, G., Barthel, S. R., 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=Analysis+of+Physiologic+E-Selectin-Mediated+Leukocyte+Rolling+on+Microvascular+Endothelium.">Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium.</a> J. Vis. Exp. (24), e1009 (2009).</li><li>Petzelbauer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+dermal+microvascular+endothelial+cell+antigen+expression+and+cytokine+responsiveness+in+situ+and+in+cell+culture.">Heterogeneity of dermal microvascular endothelial cell antigen expression and cytokine responsiveness in situ and in cell culture.</a> J. Immunol. 151, 5062-5072 (1993).</li><li>Bonder, C. S., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+leukocyte+recruitment+to+splanchnic+organs+to+reduce+inflammation.">Modulating leukocyte recruitment to splanchnic organs to reduce inflammation.</a> Am J Phys - Gastrointestinal and Liver Phys. 284, G729-G733 (2003).</li><li>Cuvelier, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eosinophil+adhesion+under+flow+conditions+activates+mechanosensitive+signaling+pathways+in+human+endothelial+cells.">Eosinophil adhesion under flow conditions activates mechanosensitive signaling pathways in human endothelial cells.</a> J. Exp. Med. 202, 865-876 (2005).</li><li>Massia, S. P., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+endothelial+cell+interactions+with+surface-coupled+adhesion+peptides+on+a+nonadhesive+glass+substrate+and+two+polymeric+biomaterials.">Human endothelial cell interactions with surface-coupled adhesion peptides on a nonadhesive glass substrate and two polymeric biomaterials.</a> J. Biomed. Mat. Res. 25, 223-242 (1991).</li></ol>

<html> Forskere har rutinemæssigt anvendt endotelceller fra forskellige kar til at studere neutrofilrekruttering og sjælevandring. Eksempler omfatter, men er ikke begrænset til, dermale mikrovaskulære endotelceller 12, lever sinusformede endotelceller 13 og endotelceller fra human navlevene 10. Blandt dem HUVEC vundet bred popularitet på grund af deres relativt lette isolering og tilgængelighed. HUVEC er en stærk in vitro modelsystem, som efterligner mange endotelceller pr...

<table border="1"><tbody><tr><td> Reagens og udstyr </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> Collagenase Type 1 </td><td> Worthington </td><td> 4197 </td><td></td></tr><tr><td> Cord buffer </td><td></td><td></td><td> Sammensætning: 140 mM NaCI 4 mM KCI 10 mM D-glucose i 1 mM NaH 2PO 4 / Na2HPO 4-puffer ved pH 7,4 </td></tr><tr><td> Endotheliale cellemedier (ECM) </td><td></td><td></td><td> M199 med eagle salte suppleret med 16% humant serum indeholdende 100 enheder penicillin 100 ug streptomycin og 0,3 mg L-glutamine/ml </td></tr><tr><td> M199 </td><td> GIBCO </td><td> 31100-035 </td><td></td></tr><tr><td> Penicillin streptomycin Glutamat (100X) </td><td> Invitrogen </td><td> 10378-016 </td><td&gt; </td></tr><tr><td> Ibidi kamre </td><td> Ibidi </td><td> 80606 </td><td></td></tr><tr><td> TNF-α </td><td> Prepro Tech </td><td> 300-01A </td><td></td></tr><tr><td> Humant albumin 20% opløsning </td><td> Gemini Bioproducts </td><td> 800120050 </td><td></td></tr><tr><td> HBSS uden Ca2 + og Mg2 + </td><td> Sigma </td><td> H2487-10X </td><td></td></tr><tr><td> HBSS med Ca 2 + og Mg2 + </td><td> Sigma </td><td> H1387-10X </td><td></td></tr></tbody></table>

isolation of human umbilical vein endothelial cells and their use in the study of neutrophil transmigration under flow conditions

Neutrofiler er de mest udbredte form af hvide blodlegemer. De udgør en væsentlig del af det medfødte immunsystem 1. Under akut inflammation, er neutrofile de første inflammatoriske celler at migrere til stedet for skaden. Rekruttering af neutrofiler til en skade site er en trinvis proces, der omfatter først, udvidelse af blodkarrene til at øge blodgennemstrømningen, for det andet, mikrovaskulære strukturelle ændringer og flygte af plasmaproteiner fra blodbanen, for det tredje, valsning, adhæsion og transmigration af neutrofil på tværs af endothelium, og den fjerde akkumulering af neutrofiler på læsionsstedet 2,3. En lang række in vivo og in vitro fremgangsmåder er udviklet til at undersøgelsen af disse processer 4. Denne metode fokuserer på neutrofil transmigration tværs af humane endotelceller. En populær metode til at undersøge de molekylære processer involveret i neutrofil transmigration udnytter menneskelige neutrophils interagerer med primære humane navlevene-endotelceller (HUVEC) 5. Neutrofil isolation er blevet beskrevet visuelt andetsteds 6, og dermed denne artikel vil vise en metode til isolering af HUVEC. Når først isoleret og dyrket til konfluens, endotelceller aktiveres resulterer i opregulering af adhæsionsmolekyler og aktivering molekyler. F.eks. Aktivering af endothelceller med cytokiner såsom TNF-a resulterer i forøget E-selectin og IL-8 ekspression 7 E-selectin medierer opsamling og valsning af neutrofiler og IL-8 medierer aktivering og faste adhæsion af neutrofiler. Efter vedhæftning neutrofile transmigrate. Transmigration kan forekomme paracellularly (gennem endotelceller junctions) eller transcellularly (gennem endotelcelle selv). I de fleste tilfælde forekommer disse interaktioner under strømningsbetingelser fundet i vaskulaturen 7,8. Den parallelle plader strømningskammeret er en almindeligt anvendt system, mimikrofoner den hydrodynamiske forskydningsspændingerne fundet in vivo og muliggør studiet af neutrofilrekruttering under strømningstilstand in vitro 9,10. Flere selskaber producerer parallelle plade strømningskamre og har hver deres fordele og ulemper. Hvis fluorescerende billeddannelse er nødvendig, glas eller en optisk lignende polymer skal anvendes. Endotelceller ikke vokser godt på glas. Her udgør en let og hurtig fremgangsmåde til fasekontrast, DIC og fluorescerende afbildning af neutrofil transmigration anvendelse af en lav mængde ibidi kanal glider fremstillet af en polymer, der understøtter den hurtige adhæsion og vækst af humane endotelceller og har optiske egenskaber, der er sammenlignelige glas. I denne fremgangsmåde blev endotelceller dyrket og stimuleret med en ibidi μslide. Neutrofiler blev indført under flowbetingelser og transmigration blev vurderet. Fluorescerende afbildning af knudepunkter aktiveres tidstro bestemmelse af omfanget af paracellulær versustranscellulære sjælevandring.

Watch this Scientific Journal Video about Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions at JoVE.com

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

I denne artikel beskrives først en procedure til isolering af humane endotelceller fra navlestrengen vener og derefter viser, hvordan man bruger disse celler til at undersøge neutrofil transmigration under strømningsforhold. Ved anvendelse af en lav-volumen strømningskammeret fremstillet af en polymer med de optiske egenskaber af glas, er levende celle fluorescerende afbildning af sjældne cellepopulationer også mulige.

Isolering af Humane navlevene-endotelceller og deres anvendelse i undersøgelsen af ​​neutrofil transmigration Under strømningsforhold

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

Research

JoVE Journal

Immunology and Infection

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Kvantitative<em&gt; In vitro</em&gt; Assay til måling neutrofiladhæsion til Aktiverede primære humane mikrovaskulære endotelceller under statiske forhold

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

En Flow Vedhæftning Assay til at studere Leucocyte Rekruttering til human hepatiske Sinusformede endotel Under betingelser for forskydningsspænding

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Human Neutrophil Flow Chamber Adhesion Assay

Human neutrofil strømningskammeret adhæsionsassay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

Real-time Imaging af endotelcellefremkaldt celle vejkryds Under Neutrofil Transmigration under fysiologiske Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In vitro og in vivo model til at studere bakteriel adhæsion til karvæggen Under strømningsforhold

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

Brug af flowcytometri at vurdere tilstanden af ​​kromatin i T-celler

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrofil Isolering og analyse for at fastslå deres rolle i lymfom Cell Følsomhed til terapeutiske midler

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions

Kvantificering af monocyt Transmigration og skum celle dannelse fra personer med kroniske betændelsestilstande

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation og In Vitro kultur af Murine og menneskelige alveolær makrofager

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro

Samtidige undersøgelse af ansættelse af monocyt delpopulationer Under Flow In Vitro

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development ...

Isolering af Humane navlevene-endotelceller og deres anvendelse i undersøgelsen af ​​neutrofil transmigration Under strømningsforhold

Isolering af Humane navlevene-endotelceller og deres anvendelse i undersøgelsen af neutrofil transmigration Under strømningsforhold