Name: measuring fast calcium fluxes in cardiomyocytes
Uploaded: 2011-11-29T13:00:00
Description: Watch this Scientific Journal Video about Measuring Fast Calcium Fluxes in Cardiomyocytes at JoVE.com

Dette arbejde blev støttet af National Institutes of Health tilskud 2R01 GM53132 og P50 GM071558.

1. Loading celler med calcium indikatorer <ol><li> Plate celler i 35mm glasbund Mattek retter (eller enhver anden glasbund visning kamre). </li><li> Hvis du bruger primære myocytes pels kamrene 1 dag før med 10μg/ml laminin. Plate hjerte myocytes (isoleret fra voksne hunde, neonatale rotter eller andre organisme) i KB buffer (K-vending Tyrode buffer 35 mmol / L HEPES, 140 mmol / l KCl, 8 mmol / L KHCO 3, 2 mmol / L af MgCl 2, og 0,4 mmol / L af KH 2 PO 4, pH 7,5) og ændr...

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Vi har udviklet en metode til at se og isolere hurtige calcium signaler i levende cardiomyocytter. Mens de eksperimentelle betingelser af disse målinger tidligere har været anvendt til andre celle-systemer 10, samt cardiomyocytter 11, brug af histogrammer og bin analyse giver mulighed for data fra mange Ca 2 + begivenheder, der skal erhverves. Hurtigere begivenheder kan let isoleres fra langsommere og modeller tilpasset til at forstå deres underliggende mekanismer. Dyrkning cardiomyoc...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> ABCD1234 </td><td></td></tr></tbody></table>

measuring fast calcium fluxes in cardiomyocytes

Cardiomyocytter har flere Ca 2 + fluxe af forskellig varighed, der arbejder sammen for at optimere funktion 1,2. Ændringer i Ca 2 + aktivitet som reaktion på ekstracellulære agenter er overvejende reguleret af phospholipase Cβ-Gα q sti lokaliseret på plasma membranen, som stimuleres af stoffer såsom acetylcholin 3,4. Vi har for nylig fundet, at plasma membran protein-domæner kaldes caveolae 5,6 kan fange aktiveres Gα q 7. Dette entrapment har den virkning at stabilisere den aktiverede tilstand Gα Q og resulterer i en forlænget Ca 2 + signaler i cardiomyocytter og andre celletyper 8. Vi har afdækket dette overraskende resultat ved at måle dynamisk calcium svar på en hurtig skala i levende cardiomyocytter. Kort fortalt, er celler fyldt med en fluorescerende Ca 2 + indikator. I vores undersøgelser har vi brugt Ca 2 + Grøn (Invitrogen, Ic.), som udviser en stigning i fluorescensen emissionsintensitet efter binding af calcium ioner. Den fluorescensintensitet er derefter registreret for at bruge en line-scan mode for en laser scanning konfokal mikroskop. Denne metode gør det muligt hurtigt at tilegne sig den tid, forløb fluorescensintensitet i pixels langs en markeret linje, der producerer flere hundrede af tid spor på mikrosekund tidsskala. Disse meget hurtige spor er flyttet ind i Excel og derefter ind i Sigmaplot til analyse, og de er sammenlignet med spor opnået for elektronisk støj, gratis farvestof, og kontroller. At dissekere Ca 2 + reaktioner af forskellige flux satser, vi udført et histogram analyse, der binned pixel intensitet med tiden. Binning giver os mulighed for at gruppere over 500 spor af scanninger og visualisere de indsamlede resultater, rumligt og tidsligt på en enkelt plot. Således kan den langsomme Ca 2 + bølger, som er vanskelige at få øje på, når de scanninger er overlejret på grund af forskellige peak placering og støj, være let ses iden binned histogrammer. Meget hurtig fluxe i den tid omfanget af målingen viser en smal fordeling af intensitet på meget kort tid skraldespande mens længere Ca 2 + bølger show binned data med en bred fordeling over længere tid skraldespande. Disse forskellige tid distributioner giver os mulighed for at dissekere timingen af Ca 2 + flusmidler i cellerne, og at fastslå deres virkning på forskellige cellulære events.

Watch this Scientific Journal Video about Measuring Fast Calcium Fluxes in Cardiomyocytes at JoVE.com

Measuring Fast Calcium Fluxes in Cardiomyocytes

Vi præsenterer en metode til at isolere hurtig (mikrosekund) calcium begivenheder fra langsommere flusmidler i levende celler ved hjælp af laser scanning konfokal mikroskopi. Metoden foranstaltninger fluorescensintensitet udsving af calcium indikatorer ved at optage linje scanninger af flere hundrede pixel i en celle. Histogram analyse giver os mulighed for at isolere tidsskalaer af forskellige calcium flux.

Måling Fast Calcium Flux i cardiomyocytter

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

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