Name: methods for the isolation, culture, and functional characterization of sinoatrial node myocytes from adult mice
Uploaded: 2016-10-23T14:00:00
Description: Watch this Scientific Journal Video about Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice at JoVE.com

We thank Dr. Christian Rickert for critical reading of the manuscript. This work was supported by a grant from the National Heart Lung and Blood Institute (R01-HL088427) to CP. EJS was supported by 5T32-AG000279 from the National Institute on Aging. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Alle dyr prosedyrer ble utført i henhold til protokoller godkjent av Institutional Animal Care og bruk komité ved University of Colorado Anschutz medisinske høyskole. Den standard protokoll nedenfor er optimalisert ved hjelp av mannlige C57BL / 6J mus på 2-3 måneders alder. 1. Forbered Løsning Aksjer og hage i forkant av eksperimenter MERK: Se Materialer tabell for nødvendig utstyr og forsyninger. <ol><li> Forbered en L hver av følgende...

<ol>
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Dette notatet presenterer detaljerte protokoller for isolering og kultur av fullt differensierte sinusknute muskelceller fra voksne mus. Isolasjon protokollen gir pålitelig spontant aktive mus Sams egnet for enten umiddelbar elektrofysiologisk analyse eller etterfølgende kultur. Lignende protokoller har blitt rapportert av mange andre grupper (se for eksempel referanser 11,12,10,13-17). Imidlertid vår protokoll for å holde voksen mus Sams in vitro bevarer den karakteristiske morfologi, spontan ak...

Pacemaker muskelceller i sinusknute av hjertet (sinoatriell muskelceller, &quot;Sams&quot;) generere spontane, rytmiske aksjonspotensialer (APS) som forplanter seg gjennom hjertemuskelen å initiere hvert hjerteslag. Eksperimenter med akutt isolert Sams fra mange arter har vært avgjørende for belysning av mekanismer som bidrar til generering av pacemakeren aktivitet. Sams er høyt spesialiserte cardiomyocytes som skiller seg vesentlig fra sine kolleger i atrial og ventrikulære myokard i form av morfologi, funksjon, og protein uttrykk. Kjennetegnet av spontane APs i Sams er en spontan depolarisering under diastolen som driver membranpotensialet til terskelen for å utløse neste AP 1,2. Denne &quot;pacemaker potensial&quot; avhenger koordinert aktivitet av mange forskjellige membran strømninger inkludert &quot;morsomme dagens&quot; (jeg f), T- og L-type kalsiumstrømmer, og natrium-kalsium leren Current (I NCX), som er drevet av Ca 2+ utgivelse fra sarkoplasmatisk retikulum 3,4. Mens akutt isolerte mus Sams er en viktig eksperimentell forberedelse for studiet av pacemaking, kan isoleringen av Sams fra mus være en krevende metode for å ta i bruk fordi den utydelig anatomi og lille størrelsen på mus SAN krever en nyansert mikrodisseksjon og den kombinerte enzymatisk og mekanisk dissosiering av cellene krever omhyggelig optimalisering. Vi gir her en detaljert video demonstrasjon av en protokoll som har blitt brukt til å isolere Sams fra voksen mus for patch clamp opptak 5-8. Så vidt vi vet, er det ingen slike visuell demonstrasjon tilgjengelig fra noen annen kilde. I tillegg er en ny fremgangsmåte demonstrert i isolert som Sams fra voksen mus kan bli opprettholdt in vitro i flere dager, for derved å tillate innføring av proteiner, genetisk kodedereporter molekyler eller RNAi via adenovirus infeksjon 9.

<table border="0" cellpadding="0" cellspacing="0" width="649">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Sylgruard/Elastomer Kit</td>
			<td style="width:167px;">Dow Corning</td>
			<td colspan="2" style="width:237px;">184 SIL ELAST KIT 0.5KG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Borosilicate 9&#34; pasteur pipettes</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td>13-678-20C</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Small, round bottomed culture tubes</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:175px;">352059</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Large, round bottomed culture tubes</td>
			<td style="width:167px;">Corning</td>
			<td style="width:175px;">14-959-11B</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Elastase</td>
			<td style="width:167px;">Worthington Biochemical</td>
			<td style="width:175px;">LS002279</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Liberase TM</td>
			<td style="width:167px;">Roche</td>
			<td style="width:175px;">5401119001</td>
			<td>&#160;Tissue dissociation solution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Heparin</td>
			<td style="width:167px;">SAGENT Pharmaceuticals&#160;</td>
			<td style="width:175px;">NDC 25021-400-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Mouse Laminin</td>
			<td style="width:167px;">Corning</td>
			<td style="width:175px;">CB-354232</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">12 mm round glass coverslips</td>
			<td style="width:167px;">Fisher&#160;</td>
			<td style="width:175px;">12-545-80</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">24-well culture plate</td>
			<td style="width:167px;">Fisher</td>
			<td style="width:175px;">08-772-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Ad-mCherry</td>
			<td style="width:167px;">Vector Biolabs</td>
			<td style="width:175px;">1767</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Ad-eGFP</td>
			<td style="width:167px;">Vector Biolabs</td>
			<td style="width:175px;">1060</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Plastic, disposable transfer pipette</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Micro scissors</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:175px;">17-467-496</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Dumont #4 Forceps</td>
			<td style="width:167px;">Roboz Instruments</td>
			<td style="width:175px;">RS-4904</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Tissue Forceps</td>
			<td style="width:167px;">Roboz Instruments</td>
			<td style="width:175px;">RS-8164</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Dissecting Iris Scissors</td>
			<td style="width:167px;">WPI, Inc.</td>
			<td style="width:175px;">501264</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Dissecting Pins</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:175px;">26002-20</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Sigma</td>
			<td>71376</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>Sigma</td>
			<td>60128</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">KH2PO4</td>
			<td>Sigma</td>
			<td>60353</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma</td>
			<td>54457</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glucose</td>
			<td>Sigma</td>
			<td>G0350500</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">CaCl2</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">taurine</td>
			<td>Sigma</td>
			<td>T0625</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BSA</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">K-glutamate</td>
			<td>Sigma</td>
			<td>G1501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">K-aspartate</td>
			<td>Sigma</td>
			<td>A6558</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">MgSO4</td>
			<td>Sigma</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">creatine</td>
			<td>Sigma</td>
			<td>C0780</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGTA</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mg-ATP</td>
			<td>Sigma</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Amphotericin-B</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:175px;">1397-89-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Isoproterenol</td>
			<td style="width:167px;">Calbiochem</td>
			<td style="width:175px;">420355</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Media199</td>
			<td>Sigma</td>
			<td style="width:175px;">M4530</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">2,3-butanedione monoxime (BDM)</td>
			<td>Sigma</td>
			<td style="width:175px;">B0753</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Fetal Bovine Serum (FBS)</td>
			<td>Sigma</td>
			<td style="width:175px;">SH30071</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td style="width:175px;">A5611</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Insulin&#160;&#160;</td>
			<td>Sigma</td>
			<td style="width:175px;">I3146</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Transferrin</td>
			<td>Sigma</td>
			<td style="width:175px;">I3146</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Selenium</td>
			<td>Sigma</td>
			<td style="width:175px;">I3146</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin</td>
			<td>GE Healthcare</td>
			<td style="width:175px;">SV30010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptomycin</td>
			<td>Hyclone</td>
			<td style="width:175px;">SV30010</td>
			<td></td>
		</tr>
	</tbody>
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methods for the isolation, culture, and functional characterization of sinoatrial node myocytes from adult mice

Sinusknute muskelceller (Sams) fungere som de naturlige pacemaker for hjertet, initiere hvert hjerteslag ved å generere spontane aksjonspotensialer (APS). Disse pacemaker APs reflektere koordinert aktivitet av mange membran strømninger og intracellulær kalsium sykling. Men de nøyaktige mekanismene som driver spontan pacemaker aktivitet i Sams forblir unnvikende. Akutt isolerte Sams er en viktig forberedelse for eksperimenter for å dissekere den molekylære grunnlaget for hjerte pacemaking. Imidlertid har utydelig anatomi, kompleks mikrodisseksjon, og pirkete enzymatiske fordøyelsen forholdene hindret utbredt bruk av akutt isolert Sams. I tillegg metodene var ikke tilgjengelig før nylig å tillate mer langsiktig kultur for Sams for protein expression studier. Her gir vi en steg-for-steg-protokollen og video demonstrasjon for isolering av Sams fra voksne mus. En fremgangsmåte er også vist for å holde voksen mus Sams in vitro og for expressipå av eksogene proteiner via adenovirus infeksjon. Akutt isolert og dyrket Sams fremstilt via disse metodene er egnet for en rekke av elektrofysiologiske og bildediagnostikk.

Protokollene er beskrevet her tidligere har blitt benyttet for å isolere spontant aktive Sams fra voksne mus som er egnet for en rekke forskjellige patch clamp studier 5-8. I tillegg protokollene tillater isolerte Sams som kan opprettholdes i kultur i opptil en uke. Genoverføring inn i de dyrkede celler kan bli oppnådd via adenoviral infeksjon 9. Resultatene som presenteres i dette avsnittet kommer fra vårt tidligere arbeid og er vist her som eksempler på hva s...

Watch this Scientific Journal Video about Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice at JoVE.com

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Metoder demonstreres for isolering av sinusknute muskelceller (SAMS) fra voksen mus for patch clamp elektrofysiologi eller bildediagnostikk. Isolerte celler kan brukes direkte eller kan holdes i kultur for å tillate ekspresjon av proteiner av interesse, slik som genetisk er kodet for pressen.

Metoder for isolering, kultur og Funksjonell karakterisering av sinusknute muskelceller fra voksne mus

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

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Biology

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