Name: measurement of antibody effects on cellular function of isolated cardiomyocytes
Uploaded: 2013-03-08T12:00:00
Description: Watch this Scientific Journal Video about Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes at JoVE.com

Dette arbejde blev støttet af Sonderforschungsbereich Transregio 19 (SFB/TR19, C2) af Deutsche Forschungsgemeinschaft (DFG), Forbundsministeriet for Økonomi og Teknologi projekt ZIM-KF 2727801MD0 og Center for Innovation Kompetence - humorale immunreaktioner i kardiovaskulære sygdomme ( ZIK-hike, BMBF FKZ 03Z2CN12) af Forbundsministeriet for Uddannelse og Forskning, Tyskland. Boliger og eksperimenter med dyr blev udført i overensstemmelse med anbefalingerne fra Society for Laboratory Animal Science (Gesellschaft für Versuchstierkunde, GV-SOLAS) og Federation of European Laboratory Animal Science Association (FELASA).

1. IgG Isolering fra patientprøver <ol><li> Forbered mini-immunoadsorption kolonner ved at udfylde 3 ml anti-IgG sepharose per 2 ml patient EDTA plasma ind i en tom Econo Pac kolonne. IgG isolation kræver mindst 2 ml af patientens plasma. </li><li> Placere et filter på toppen af ​​anti-IgG sepharose, skar nedre spids af kolonnen og tryk på filteret for at nå en strømningshastighed på cirka 1 dråbe / sek. Lad bevarelse opløsning løber ud af søjlen. </li><li> Søjlen vaskes med 3 volumener 0,9% NaCI pr m?...

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Den fremlagte metode giver en passende måde at opdage funktionelt effektive hjerte autoantistoffer hos patienter med DCM af uklar oprindelse. I sammenligning med andre metoder, påvisning af funktionelle aktive antistoffer mod β1-adrenoceptor fx ved deres virkning på cAMP-niveauer 6, vores metode er uafhængig af en specifik epitop. Selvfølgelig er der andre epitop uafhængige analyser er beskrevet i litteraturen, dvs tælle slaghastighed af neonatale cardiomyocytter 13, der m...

<table border="1"><tbody><tr><td> Navn på reagens / udstyr </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Immunosorba konserveringsopløsning </td><td> Fresenius Medical Care </td><td> 903609211 </td><td></td></tr><tr><td> Collagenase type 2 </td><td> Cellesystem </td><td> LS004176 </td><td></td></tr><tr><td> BSA, fedtsyrefrit </td><td> Sigma-Aldrich </td><td> A6003 </td><td></td></tr><tr><td> Laminin </td><td> Sigma-Aldrich </td><td> L2020 </td><td></td></tr><tr><td> Fura-2:00 </td><td> Sigma-Aldrich </td><td> F0888 </td><td> 1 mg / ml i DMSO </td></tr><tr><td> Økono Pac kromatografisøjler </td><td> BioRad </td><td> 732-1010 </td><td></td></tr><tr><td> Spectra / Por Biotech Cellulose Ester Dialyse Membrane </td><td> Spectrumlabs </td><td> 131.417 </td><td></td></tr><tr><td> 4 godt Chambered dækglas </td><td> Nunc </td><td> 155.383 </td><td></td></tr><tr><td> Myocyt Calcium og kontraktilitet Recording System </td><td> IonOptix </td><td></td><td></td></tr><tr><td> IonWizard 6,0 ​​analyse software </td><td> IonOptix </td><td></td><td></td></tr></tbody></table>

measurement of antibody effects on cellular function of isolated cardiomyocytes

Dilateret kardiomyopati (DCM) er en af de vigtigste årsager til hjertesvigt hos yngre voksne 1. Selv om genetiske disposition og redegørelse for giftige stoffer er kendte årsager til denne sygdom i omkring en tredjedel af patienterne, oprindelsen af ​​DCM er fortsat stort set uklar. I et betydeligt antal af disse patienter har autoantistoffer mod hjerte-epitoper blevet opdaget og er mistænkt for at spille en central rolle i opståen og progression af sygdommen 2,3. Betydningen af hjerte-autoantistoffer understreges af en hæmodynamisk bedring hos DCM patienter efter eliminering af autoantistoffer ved immunoadsorption 3-5. En række specifikke antigener er allerede blevet identificeret 2,3 og antistoffer mod disse mål kan påvises ved immunoassays. Imidlertid kan disse analyser ikke diskriminere mellem stimulere (og derfor funktionelt effektive) og blokere autoantistoffer. Der er stigende evidence, at denne skelnen er afgørende 6,7. Det kan også antages, at de mål for en række cardiotropic antistoffer er stadig ukendt og kan derfor ikke påvises ved immunoassays. Derfor har vi etableret en fremgangsmåde til påvisning af funktionelt aktive cardiotropic antistoffer, der er uafhængige af deres respektive antigen. Baggrunden for fremgangsmåden er den høje homologi sædvanligvis observeret for funktionelle regioner af kardiale proteiner i mellem pattedyr 8,9. Dette antyder, at hjerte-antistoffer rettet mod humane antigener vil krydsreagere med ikke-humane målceller, hvilket tillader test af IgG fra DCM patienter på voksne rotter cardiomyocytes. Vores fremgangsmåde består af tre trin: først bliver IgG isoleret fra patientplasma med Sepharose koblet anti-IgG-antistoffer opnået fra immunoadsorption søjler (PlasmaSelect, Teterow, Tyskland). For det andet voksne kardiomyocytter isoleres ved collagenaseperfusion i en Langendorff perfusion apparat under anvendelse af aprotocol modificeret fra tidligere arbejder 10,11. De opnåede kardiomyocytter er fastgjort til laminin-overtrukne kamre dækglasset og farvet med Fura-2, en calcium-selektivt fluorescerende farvestof, som let kan bringes ind i cellen at observere intracellulært calcium (Ca2 +) indhold 12. I det sidste trin, virkningen af patientens IgG på cellens afkortning og Ca2 +-transienter i felt stimulerede cardiomyocytes overvåges online ved hjælp af et kommercielt myocyt calcium og kontraktilitet overvågningssystem (IonOptix, Milton, MA, USA) forbundet til en standard omvendt fluorescerende mikroskop.

To eksempler til måling af cellulær inotropi i felt stimuleret voksne kardiomyocytter (fig. 2) ved hjælp af en myocyt Ca2 + og kontraktilitet registreringssystem er angivet nedenfor. Figur 3 giver et indtryk af en kontrolmåling, mens der i figur 4 virkningen af cardiodepressive antistoffer af en patient med DCM vises. I begge eksempler, initial celle afkortning og Ca 2 + forbigående under superfusionsapparatet med ...

Watch this Scientific Journal Video about Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes at JoVE.com

Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

I denne procedure giver det muligt at opdage funktionelle effektive cardiotropic autoantistoffer i plasmaet hos patienter med dilateret cardiomyopati, uanset det specifikke antigen, ved at analysere virkningen af ​​isolerede patient immunoglobulin på cellulær afkortning og intracellulært calcium transienter i isolerede rotte cardiomyocytes.

Måling af antistof Virkninger på cellulær funktion af isolerede cardiomyocytes

Dilated cardiomyopathy  (DCM) is one of the main causes for heart failure in younger adults1.  Although genetic disposition and exposition to toxic ...

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