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 arbeidet ble støttet av UCSF Institutt for anestesi og Perioperative Care.

En. Platekledning og vedlikehold av mikrovaskulær endotelceller <ol><li> Tine og vokse din mikrovaskulære endotelceller i henhold til produsentene følger instruksjonene. I denne protokollen, ble cellene dyrket i EGM-2 medium MV (Lonza), og det anbefales at alle eksperimenter utføres i løpet av to uker etter opptining for å redusere mulig variasjon på grunn av passasje nummer. Våre eksperimenter med HMVEC-Lung utføres mellom passasjer 4-9. </li><li> Dag 1: Når cellene nå 80-90% confluency, trypsinize og resu...

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De mest kritiske trinn for en vellykket neutrofil / mikrovaskulær endotelcelle-adhesjon analysen er: 1) Anvendelse av lav passasje nummer (&lt;9), sunne endotelceller, 2) opprettholdelse av de isolerte nøytrofiler ved en lav tetthet (dvs. &lt;5 x 10 celler 6 / ml) og bruker dem innen to timer med isolasjon, og 3) Smakfullt vask av Calcein AM-merket nøytrofile og bruk av Calcein AM-merket nøytrofile i tide for å minimere EC forurensning og tap av Calcein fra de nøytrofile, henholdsvis . <p c...

Siden det vaskulære endotel er i direkte kontakt med det sirkulerende blod, er det en unik plassert for å initiere en hurtig inflammatorisk respons under infeksjon eller skade. Endotelceller (ECS) uttrykte mønstergjenkjenning reseptorer som gjenkjenner en rekke bevarte bakterielle komponenter og fare molekyler, og reseptorer for vert inflammatoriske mediatorer som TNF-alfa. Aktivering av disse reseptorene ECS å utskille cytokiner (for eksempel IL-6, IL-8, CXCL1 og CCL2), og å oppregulere adhesjonsmolekyler (f.eks E / P-selektin, VCAM-1 og ICAM-1) på deres celleoverflate ett , 2.. Disse molekylene alt til rette for lokalisering av leukocytter til nettsteder for infeksjon og skade for å fjerne vert for smittestoffer og initiere vev reparasjon 3,4. Den nøytrofile responsen på infeksjon innebærer en godt koordinert samspill mellom blodåreendotelets og svarer nøytrofile. Ved EF-aktivering, IL-8 utskilles og danner en intravaskulær gradient på endotelet som gjør at nøytrofile hjemme på stedet for infeksjon eller skade 5,6. E / P-selectins megle nøytrofile fangst og rulle gjennom relativt svake assosiasjoner med glycomolecules på nøytrofile celleoverflaten. Disse interaksjonene, sammen med IL-8 binding til sine beslektede reseptorer, lette robust, integrinantistoff-mediert vedlegg og eventuell arrestasjon av nøytrofile på endotelceller overflaten 7-10. Etter arrestasjonen, kan nøytrofile migrere ut av blodkar til de spesifikke områder av smitte til direkte eliminere patogener, generere nøytrofile ekstracellulære feller for å hindre spredning av patogener, fremme sårheling og utgi flere faktorer som rekrutterer andre leukocytter som monocytter, makrofager og dendrittiske celler 11-17. Beskrevet i denne rapporten er en in vitro metode for å kvantifisere nøytrofile tilslutning til microVascular egenkapitalbevis etter aktivering av verten inflammatorisk megler TNF-alfa. Denne analysen er laget for å vurdere aktivering av egenkapitalbevis, og ikke nøytrofile. Primære humane neutrofiler er først isolert ved hjelp av densitetsgradient separasjon, og blir deretter merket med Calcein acetoksymetyl (AM). Esterasene innenfor levende celler hydrolyze Calcein AM til den svært fluorescerende Calcein molekyl med en eksitasjon av 492-495 nm og utslipp av 513-516 nm 18. De fluorescently-merket nøytrofile blir deretter inkubert med EF monolagene, og ikke-heftende nøytrofile senere blir fjernet. Fluorescensen av de gjenværende, bundne nøytrofiler blir så målt ved hjelp av et fluorescens-spektrofotometer, og beregnes som en prosent av total neutrofil fluorescens inndata per brønn. Denne metode har den ytterligere fordel at de bundne Calcein-merkede nøytrofiler som brukes i spektrofotometri direkte kan visualiseres ved hjelp av fluorescens-mikroskopi for å gi en mer kvalitativ lest ut av EC acaktiverings. Siden denne analysen er utført under statiske forhold, vil bare de aller første hendelser som oppstår i nøytrofile vedheft kaskade bli vurdert. Dette bekreftes i denne rapporten bruker E-selektinantistoffer blokkerende antistoffer for å vise at nøytrofile tilslutning til TNF-alfa-behandlet menneskelig lunge microvascular EC (HMVEC-Lung) monolagene er drastisk redusert når samspillet med E-selectin blir forstyrret. I tillegg til TNF-alfa, har vi med hell brukt denne analysen for å fastslå omfanget av menneskelig umbilical vein EC (HUVEC) aktivering av Toll-like receptor 1/2 agonister peptidoglycan-assosiert lipoprotein (PAL), murein lipoprotein (MLP) og Pam3Cys og HMVEC-Lung aktivering av Pam3Cys 19,20. I tillegg har vi med hell brukt denne analysen med kinase hemmere og etter RNAi-mediert knockdown av overflaten og cytoplasmatiske proteiner i HMVEC-Lung, som tyder på at denne metodikken er kompatibel med en rekke biokjemiske og screening-analyser 20. Oppsummert gir denne analysen en lett å bruke, reproduserbar, mer funksjonell måte å få tilgang til omfanget av EC aktivering av 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

Blodåreendotelets spiller en vesentlig rolle i den inflammatoriske respons. I den akutte fasen av betennelse, er endotelceller (ECS) aktiveres av vert meklere eller direkte av konserverte mikrobielle komponenter eller host-avledet fare molekyler. Aktiverte egenkapitalbevis uttrykke cytokiner, chemokiner og adhesjonsmolekyler som mobiliserer, aktivere og beholde leukocytter på stedet av infeksjon eller skade. Nøytrofile granulocytter er de første leukocytter å ankomme, og holder seg til endotelet gjennom en rekke adhesjonsmolekyler tilstede på overflaten av begge celler. De viktigste funksjonene til nøytrofile er å direkte eliminere mikrobielle trusler, fremme rekruttering av andre leukocytter gjennom utgivelsen av flere faktorer, og initiere sår reparasjon. Derfor er rekruttering og vedlegg til endotelet et kritisk punkt i initiering av den inflammatoriske respons. I denne rapporten beskriver vi en in vitro nøytrofile vedheft analysen hjelpCalcein AM-merket primære humane neutrofiler til å kvantifisere omfanget av mikrovaskulær endotelial celleaktivering under statiske forhold. Denne metode har den ytterligere fordel at de samme prøvene kvantifisert ved fluoresens-spektrofotometri kan også visualiseres direkte ved hjelp av fluorescens-mikroskopi for en mer kvalitativ vurdering av nøytrofil binding.

For å oppnå pålitelige, reproduserbare resultater ved hjelp av en neutrofil bindingsanalyse, er det viktig at helse og sammenflyting av de mikrovaskulære endotelceller er optimal på dagen for analysen, som illustrert med HMVEC-Lung i figur 1.. I tillegg er det viktig at lav passasje antall microvascular egenkapitalbevis brukes (dvs. mindre enn ni passasjer), og følgelig anbefaler vi utfører alle eksperimenter innen to uker etter tining. Helsen til neutrofiler er også av største betydni...

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

Nøytrofile tilslutning til aktivert endotel på nettstedene til infeksjon er en integrert del av vertens inflammatorisk respons. Beskrevet i denne rapporten er en nøytrofile bindingsanalyse som gir mulighet for<em&gt; In vitro</em&gt; Kvantifisering av primær menneskelig nøytrofile binding til endotelceller aktiveres av inflammatoriske mediatorer under statiske forhold.

Kvantitativ<em&gt; In vitro</em&gt; Analysen for å måle Neutrophil vedheft til Aktiverte primære humane mikrovaskulær endotelceller under statiske betingelser

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 av menneskelige umbilical vein endotelceller og deres bruk i studiet av Neutrofil Sjelevandring 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; Co-infeksjon modell for å studere<em&gt; Plasmodium falciparum</em&gt;-HIV-1 interaksjoner i Human Primary monocytt-deriverte 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

Introduksjon Kokulturanalyse for å vurdere patogenindusert nøytrofil transepiteriemigrasjon

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 Adhesion analysen til Study leucocyte Rekruttering til humane Sinus endotelet under forhold med Skjær Stress

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

Menneskelig Neutrophil Flow Chamber Adhesion analysen

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

Statisk feste analysen for Study of inte Activation 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 av endotelceller-celle Junctions Under Neutrophil Sjele 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 modell for å studere bakteriell adhesjon til fartøyet veggen 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 av Adhesjon Under skjærspenning for studier av T-lymfocytter-adhesjonsmolekyl Interactions

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