Name: quantitative in vitro assay to measure neutrophil adhesion to activated primary human microvascular endothelial cells under static conditions
Uploaded: 2013-08-23T12:00:00
Description: Watch this Scientific Journal Video about Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions at JoVE.com

Dette arbejde blev støttet af UCSF Institut for Anæstesi og Perioperative Care.

1.. Plating og vedligeholdelse af Mikrovaskulære endotelceller <ol><li> Tø og dyrke dine mikrovaskulære endotelceller ifølge producenterne leverede anvisninger. I denne protokol blev celler dyrket i EGM-2 MV medier (Lonza), og det anbefales, at alle forsøg udføres inden for to uger efter optøning for at minimere enhver variation som følge passage nummer. Vores eksperimenter med HMVEC-Lung gennemføres mellem passagerne 4-9. </li><li> Dag 1: Når cellerne når 80-90% konfluens Trypsinisér og resuspender på 12...

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De mest kritiske trin for en vellykket neutrofil / mikrovaskulære endotel celleadhæsionsassay er: 1) Anvendelse af lav passage nummer (&lt;9), sunde endotelceller, 2) Opretholdelse af de isolerede neutrofiler ved en lav densitet (dvs. &lt;5 x 10 6 celler / ml) og bruge dem inden for to timer isolation, og 3) Udsøgt vask af calcein AM-mærkede neutrofiler og brug af calcein AM-mærkede neutrofiler i tide for at minimere EF forurening og tab af calcein fra neutrofiler hhv . <p class="jove_conten...

Da det vaskulære endotel er i direkte kontakt med det cirkulerende blod, er det unikt beliggende at indlede en hurtig inflammatorisk respons under infektion eller skade. Endotelceller (EC&#39;er) udtrykkelige mønstergenkendelse receptorer, der genkender en række bevarede bakterielle komponenter og fare molekyler og receptorer for vært inflammatoriske mediatorer såsom TNF. Aktivering af disse receptorer inducerer EC&#39;er at udskille cytokiner (f.eks IL-6, IL-8, CXCL1 og CCL2), og at opregulere adhæsionsmolekyler (fx E / P-selectin, VCAM-1 og ICAM-1) på deres celleoverflade 1 , 2.. Disse molekyler alle lette lokaliseringen af leukocytter til steder for infektion og skade for at rydde vært for smitstoffer og igangsætte væv reparation 3,4. Den neutrofile respons på infektion indebærer en velkoordineret samspil mellem det vaskulære endotel og reagerer neutrofiler. Ved EF aktivering, IL-8 udskilles og danner en intravaskulær gradient på endotelet, der tillader neutrofiler til hjem til stedet for infektion eller skade 5,6. E / P-selectiner mægle neutrofil indfangning og rullende gennem relativt svage associationer glycomolecules på neutrofile celleoverfladen. Disse interaktioner, sammen med IL-8 binding til dets kognate receptorer, lette robuste, integrin-medieret binding og eventuel anholdelse af neutrofiler på endotelcelleoverfladen 7-10. Efter anholdelse, kan neutrofile migrere ud af karrene til de specifikke steder for smitte til direkte at eliminere patogener, generere neutrofile ekstracellulære fælder for at forhindre spredning af patogener, fremme sårheling og frigøre yderligere faktorer, som rekrutterer andre leukocytter såsom monocytter, makrofager og dendritiske celler 11-17. Beskrevet i denne rapport er en in vitro metode til at kvantificere neutrofil overholdelse microVascular EC&#39;er efter aktivering af værten inflammatorisk mediator TNF. Denne analyse er at vurdere aktiveringen af ​​ECS og ikke neutrofiler. Primære humane neutrofiler er først isoleret ved anvendelse densitetsgradient separation, og derefter mærket med calcein acetoxymethyl (AM). Esteraser i levende celler hydrolysere calcein AM til den meget fluorescerende calcein molekyle med et excitation af 492-495 nm og emission af 513-516 nm 18 år. De fluorescens-mærkede neutrofiler inkuberes derefter med EF-monolag, og ikke-adhærente neutrofiler efterfølgende fjernet. Fluorescensen af ​​de resterende, bundne neutrofiler måles derefter ved hjælp af et fluorescensspektrofotometer, og beregnes som en procent af den samlede neutrofil fluorescens input per brønd. Denne metode har den yderligere fordel, at de bundne calcein-mærkede neutrofiler anvendes i spektrofotometri kan visualiseres direkte ved hjælp af fluorescensmikroskopi for at give en mere kvalitativ udlæses af EF acvation. Da dette assay udføres under statiske betingelser, vil kun de meget indledende begivenheder, der opstår i neutrofiladhæsion kaskade vurderes. Dette bekræftes i denne rapport ved hjælp E-selectin blokerende antistoffer at vise, at neutrofil overholdelse TNF-behandlet human lunge mikrovaskulære EC (HMVEC-Lung) monolag er drastisk reduceret, når samspillet med E-selectin er forstyrret. Ud over TNFa, har vi med succes anvendt dette assay til at bestemme omfanget af menneskers navlevene EF (HUVEC) aktivering af Toll-like receptor 1/2 agonister peptidoglycan-associeret lipoprotein (PAL), murein lipoprotein (MLP) og Pam3Cys og HMVEC-Lunge aktivering med Pam3Cys 19,20. Desuden, vi succesfuldt anvendt dette assay med kinaseinhibitorer og efter RNAi-medieret knockdown af overflade-og cytoplasmiske proteiner i HMVEC-Lung, antyder, at denne metode er forenelig med en række biokemiske og screening assays 20. Sammenfattet indeholder denne analyse en nem at bruge, reproducerbar, mere funktionel måde at få adgang omfanget af EF-aktivering ved inflammatoriske mediatorer in vitro.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>HMVEC-Lung</td>
 <td>Lonza</td>
 <td>CC-2527</td>
 <td></td>
 </tr>
 <tr>
 <td>EGM-2 MV</td>
 <td>Lonza</td>
 <td>CC-3202</td>
 <td></td>
 </tr>
 <tr>
 <td>HBSS</td>
 <td>Life Technologies</td>
 <td>14175-095</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>48-well Tissue Culture Plates</td>
 <td>BD Falcon</td>
 <td>353078</td>
 <td></td>
 </tr>
 <tr>
 <td>PBS (without phenol red)</td>
 <td>UCSF Cell Culture Facility</td>
 <td>CCFAL001 </td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>D-PBS (without Ca2+ and Mg2+ and phenol red)</td>
 <td>UCSF Cell Culture Facility</td>
 <td>CCFAL003</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>RPMI-1640 (without phenol red)</td>
 <td>Life Technologies</td>
 <td>11835-030</td>
 <td></td>
 </tr>
 <tr>
 <td>Polymorphprep</td>
 <td>Axis-Shield</td>
 <td>1114683</td>
 <td></td>
 </tr>
 <tr>
 <td>calcein AM</td>
 <td>Life Technologies</td>
 <td>C3099</td>
 <td>(0.995 mM) stock soln</td>
 </tr>
 <tr>
 <td>Trypan Blue Solution</td>
 <td>Sigma-Aldrich</td>
 <td>T8154</td>
 <td></td>
 </tr>
 <tr>
 <td>40 &mu;M filters</td>
 <td>VWR</td>
 <td>21008-949</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>FLUOstar OPTIMA Spectrophotometer</td>
 <td>BMG LABTECH</td>
 <td>-</td>
 <td></td>
 </tr>
 </tbody>
</table>

quantitative in vitro assay to measure neutrophil adhesion to activated primary human microvascular endothelial cells under static conditions

Det vaskulære endotel spiller en integrerende del i det inflammatoriske respons. Under den akutte fase af inflammation, der endotelceller (EC&#39;er) aktiveres ved værts mediatorer eller direkte af konserverede mikrobielle komponenter eller værtsafledte fare molekyler. Aktiverede ECS udtrykke cytokiner, kemokiner og adhæsionsmolekyler, der mobiliserer, aktivere og fastholde leukocytter på stedet for infektion eller skade. Neutrofiler er de første leukocytter at ankomme, og klæbe til endotelet gennem en række adhæsionsmolekyler stede på overfladerne af begge celler. De vigtigste funktioner i neutrofiler er direkte eliminere mikrobielle trusler fremme af ansættelse af andre leukocytter gennem frigivelse af yderligere faktorer, og igangsætte sårheling. Derfor er deres rekruttering og tilknytningen til endotel er et afgørende skridt i indledningen af ​​den inflammatoriske respons. I denne rapport beskriver vi en in vitro neutrofiladhæsion assay hjælpcalcein AM-mærkede primære humane neutrofiler at kvantificere omfanget af mikrovaskulære endotelcelle aktivering under statiske forhold. Denne metode har den yderligere fordel, at de samme prøver kvantificeret ved fluorescens-spektrofotometri også kan visualiseres direkte ved hjælp af fluorescensmikroskopi for en mere kvalitativ vurdering af neutrofil binding.

For at opnå pålidelige, reproducerbare resultater ved hjælp af en neutrofil bindingsassay, er det vigtigt, at sundhed og sammenflydning af de mikrovaskulære endotelceller er optimale på dagen for assayet, som illustreret med HMVEC-Lung i figur 1. Desuden er det bydende nødvendigt, at lave passage nummer mikrovaskulære ECS brugt (dvs. mindre end 9 passager), og derfor anbefaler vi at udføre alle eksperimenter inden for to uger efter optøning. Sundheden for neutrofile er også af allerst...

Watch this Scientific Journal Video about Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions at JoVE.com

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

Neutrofil tilslutning til den aktiverede endotel på steder med infektion er en integreret del af værtens inflammatoriske respons. Beskrevet i denne rapport er en neutrofil binding assay, der giver mulighed for<em&gt; In vitro</em&gt; Kvantificering af primær human neutrofil binding til endotelceller aktiveres af inflammatoriske mediatorer under statiske betingelser.

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 ...

Research

JoVE Journal

Immunology and Infection

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

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

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

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, ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

En<em&gt; In vitro</em&gt; Samtidig infektion model for at studere<em&gt; Plasmodium falciparum</em&gt;-HIV-1 Interactions i humane primære Monocytafledte immunceller

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

In vitro Coculture Assay at vurdere Patogen induceret Neutrophil Trans-epitel Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

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 ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

Static adhæsionsassayet for Studiet af Integrin Aktivering i T-lymfocytter

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

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 ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Analyse af Vedhæftning Under Shear Stress for Studiet af T-lymfocyt-adhæsionsmolekyle Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...