Name: isolation and culture of adult mouse cardiomyocytes for cell signaling and in vitro cardiac hypertrophy
Uploaded: 2014-05-21T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy at JoVE.com

Dette arbejde blev støttet af National Heart, Lung, og Blood Institute Grant HL-36573. Vi takker David Sowa til at redigere dette manuskript.

Al forskning på mus blev udført i henhold til procedurer og retningslinjer for National Institutes of Health, og de protokoller, blev godkendt af Institutional Animal Care og brug Udvalg University of Toledo, College of Medicine og Life Sciences. 1.. Perfusionssystem Forberedelse <ol><li> Dagen før isolation, udfylde hele perfusion (herunder reservoirer) med en opløsning af hurtig alkalisk rester vaskemiddel. Lad opløsningen suge ind i systemet i et par timer eller natten over. </li><...

<ol>
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For den bedste forberedelse, de mest kritiske trin er: 1) omgående at tilslutte musen hjerte til kanylen efter udskæring; 2) passende hjerte perfusion. Bemærk også, at vandkvalitet, perfusion temperatur, pH buffer, sterilisering, kemisk renhed, og ren, ikke-forurenet slanger og kamre er også vigtige faktorer. 18,2 MOhm molekylærbiologi-grade H2O er stærkt anbefales til forberedelse buffer. PH-værdien af ​​200 mM ATP stamopløsning bør justeres til 7,2, et skridt, som er let at gå glip af. Det er vigti...

Cardiomyocytter ikke proliferativ. Der er nogle atrielle cardiomyocyte cellelinjer, som HL-1 og AT-1-celler afledt fra mus atrielle tumorer; men der er ingen voksne ventrikulære cardiomyocyte cellelinjer til rådighed for forskningen. Primære cellekulturer af voksne mus cardiomyocytes giver en kraftfuld model for hjerte forskning på de cellulære og molekylære niveau. Til dato har de været brugt i udstrakt grad til biokemiske, fysiologiske og farmakologiske forskning 1.. Derudover har den hyppige brug af genetisk modificerede mus nødvendiggjort effektive metoder til cardiomyocyte isolation. Renkultur giver mulighed for betingelser fri interaktion med andre organer og den systemiske cirkulation, såsom gennem endogene neurohormonal og hormon-lignende faktorer 2,3. Vellykket isolering af cardiomyocytes Dog kan være udfordrende. Protokollen vi indfører heri er baseret på vores erfaringer med voksne rotter cardiomyocytes og beskrevet af O&#39;Connell et al 4,5 metode. Især i 2007 5, de beskrev detaljerede teknikker fra buffer præparater til cellekultur og funktionel analyse. Da mus myocytes er meget modtagelige for kontraktur forhold til rotte cardiomyocytes er de buffere eller medier, der anvendes til perfusion, fordøjelse, Ca 2 + tolerance, plating og kultur i muse-protokol suppleret med en uspecifik excitation-kontraktion kobling inhibitor, 2, 3-butandion monoxime (BDM) for at hæmme deres spontane sammentrækning, dermed levedygtighed og udbyttet af stavformede myocytes forbedres markant. I protokollen indføres her, er myocytterne separeret i en høj kalium buffer fra den isolerede hjerte ved modificeret Langendorff perfusion med type II collagenase. Collagenase II effektivt nedbryder den intercellulære matrix og frigiver cellerne. Perfusionsopløsningen holder også cellulær metabolisme på et lavt niveau 6. I additipå den isolerede hjerte system fra Harvard Apparatus vi brugt her er godt designet til præcis temperatur og konstant tryk kontrol 7. Denne fremgangsmåde giver meget reproducerbare præparater og ensartede populationer af enkelt celletype, som kan anvendes i dyrkning natten over til måling signalproteiner eller 2-3 dages kultur for hjerte hypertrofiske assays.

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isolation and culture of adult mouse cardiomyocytes for cell signaling and in vitro cardiac hypertrophy

Teknologiske fremskridt har gjort genmodificerede mus, herunder transgene og gen-knockout-mus, et vigtigt redskab i mange forskningsområder. Voksen cardiomyocytes er bredt accepteret som en god model for hjerte-cellulær fysiologi og patofysiologi samt til farmaceutisk intervention. Genetisk modificerede mus udelukker behovet for komplicerede cardiomyocyte infektion processer til at generere den ønskede genotype, som er ineffektive på grund af cardiomyocytter &#39;terminal differentiering. Isolering og dyrkning af høj kvantitet og kvalitet funktionelle cardiomyocytes vil dramatisk gavne hjerte-kar-forskning og give et vigtigt redskab til celle signalering transduktion forskning og udvikling af lægemidler. Her beskriver vi en veletableret metode til isolering af voksne mus cardiomyocytes, der kan gennemføres med lidt træning. Musen hjerte udskåret og kanyle til en isoleret hjerte-system, og derefter perfunderet med en calcium-fri og høj potassium puffer efterfulgt af type II collagenasefordøjelse i Langendorff retrograd perfusion mode. Denne protokol giver et ensartet resultat til indsamling af funktionelle voksen mus cardiomyocytter fra en række af genetisk modificerede mus.

1.. Vellykket Isolation Kvantificering To kriterier anvendes til at kvantificere succes isolation: første, det samlede antal cardiomyocytes isolerede, og for det andet forholdet mellem stavformede calcium-tolerant at runde ikke-tolerante myocytes. Generelt dette protokol tager omkring 75-90 minutter fra hjertet fjernelse til myocyt yderklædningen og udbytter omkring 1 million stavformede cardiomyocytes (70-90% af den samlede myocytes høstet) fra en voksen mus hjerte. Dette kan variere med mu...

Watch this Scientific Journal Video about Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy at JoVE.com

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Isolering og dyrkning af Voksen Mouse kardiomyocytter for cellesignalering og<em&gt; In vitro</em&gt; Hjertehypertrofi

Vi beskriver en pålidelig metode til isolering af voksne mus cardiomyocytter. Denne protokol giver et ensartet resultat til dyrkning af funktionelle voksne kardiomyocytter fra en række af genetisk modificerede mus.

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

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Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

Single Cell transkriptionel Profilering af Voksen Mouse cardiomyocytter

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Isolering og dyrkning af individuelle myofibre og deres satellit celler fra voksne skeletmuskler

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Isolering og dyrkning af Neonatal Mouse cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

Isolation, Kultur og funktionel karakterisering af Voksen Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Isolering og Fysiologisk Analyse af Mouse cardiomyocytter

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

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Isolering og dyrkning af Dental Epiteliale stamceller fra voksne mus Fortand

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

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Isolation, Kultur og transduktion af Voksen Mouse cardiomyocytter

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

Isolering og kultur af primære muskeratinocytter fra neonatal og voksen mus hud

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

Isolering af mus epidermal keratinocytter og deres in vitro-Clonogen kultur

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately ...