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 dyreforsøg blev udført i overensstemmelse med protokoller, der er godkendt af Institutional Animal Care og brug Udvalg fra University of Colorado Anschutz Medical Campus. Standardprotokollen nedenfor er blevet optimeret ved hjælp C57BL / 6J mus på 2-3 måneder gamle. 1. Forbered Løsning Aktier og forsyninger i Advance forsøgsplanlægning BEMÆRK: Se Materialer Table for nødvendigt udstyr og forsyninger. <ol><li> Forbered 1 L hver af...

<ol>
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Denne artikel præsenterer detaljerede protokoller for isolering og dyrkning af fuldt differentierede sinoatriale node myocytter fra voksne mus. Isoleringen protokol pålideligt producerer spontant aktive mus SAM egnede til enten øjeblikkelig elektrofysiologisk analyse eller efterfølgende kultur. Lignende protokoller er blevet rapporteret af mange andre grupper (f.eks se referencer 11,12,10,13-17). Men vores protokol for opretholdelse voksne mus SAM in vitro bevarer den karakteristiske morfologi, s...

Pacemaker myocytter i sinoatrialt knude af hjertet (sinoatriale myocytter, &quot;SAM&quot;) generere spontane, rytmiske aktionspotentialer (APS), der udbreder gennem myokardiet at indlede hvert hjerteslag. Forsøg under anvendelse akut isoleret SAM fra mange arter har været afgørende for belysning af mekanismer, der bidrager til frembringelsen af ​​pacemakeren aktivitet. SAM er højt specialiserede cardiomyocytter der afviger væsentligt fra deres modparter i atrial og ventrikulær myocardium i form af morfologi, funktion og protein udtryk. Kendetegnet for spontane APs i SAM er en spontan depolarisering under diastole, der driver membranpotentialet til tærsklen for at udløse den næste AP 1,2. Denne &quot;pacemaker potentiale&quot; afhænger af koordineret aktivitet af mange forskellige membran strømninger herunder &quot;funny nuværende&quot; (I f), T- og L-type calcium strømninger, og natrium-calcium veksler current (I NCX), som drives af Ca2 + frigivelse fra reticulum 3,4. Mens akut isoleret muse SAM&#39;er er en væsentlig eksperimentel forberedelse til studiet af pacemaking, kan isoleringen af ​​SAM fra mus være en udfordrende metode til at vedtage fordi utydelig anatomi og lille størrelse af muse SAN kræver en nuanceret mikrodissektion og den kombinerede enzymatiske og mekaniske dissociation af cellerne kræver omhyggelig optimering. Vi giver her en detaljeret video demonstration af en protokol, der er blevet brugt til at isolere SAM fra voksne mus for patch clamp optagelser 5-8. Til vores viden, er der ikke sådan visuel demonstration fra nogen anden kilde. Desuden er en ny metode demonstreret ved hvilken isoleret SAM fra voksne mus kan opretholdes in vitro i adskillige dage, hvilket tillader indførelsen af proteiner, genetisk indkodedereportermolekyler eller RNAi via adenoviral infektion 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>
</table>

methods for the isolation, culture, and functional characterization of sinoatrial node myocytes from adult mice

Sinusknude myocytter (SAM) fungere som de naturlige pacemaker i hjertet, indleder hvert hjerteslag ved at generere spontane aktionspotentialer (APS). Disse pacemaker APs afspejler koordineret aktivitet af talrige membranstrømme og intracellulært calcium cykling. Men de præcise mekanismer, der driver spontan pacemaker aktivitet i SAM forblive undvigende. Akut isolerede SAM er en væsentlig forberedelse til eksperimenter for at dissekere den molekylære basis for cardiac pacemaking. Imidlertid har den utydelige anatomi, kompleks mikrodissektion, og pedantisk enzymatiske fordøjelse betingelser forhindret udbredt anvendelse af akut isolerede SAM. Derudover metoder var ikke tilgængelige indtil for nylig at tillade mere langsigtet kultur af SAM til proteinekspression studier. Her giver vi en trin-for-trin-protokol og video demonstration til isolering af SAM fra voksne mus. Fremgangsmåde demonstreres også for opretholdelse voksne mus SAM in vitro og for Express godtpå af eksogene proteiner via adenoviral infektion. Akut isoleret og dyrket SAM fremstillet via disse metoder er egnede til en række forskellige elektrofysiologiske og billeddannende undersøgelser.

De her beskrevne protokoller har tidligere været anvendt til at isolere spontant aktive SAM fra voksne mus, som er egnet til en række forskellige patch clamp-forsøg 5-8. Desuden protokollerne tillader isolerede SAM&#39;er, som kan opretholdes i kultur i op til en uge. Genoverførsel til de dyrkede celler kan opnås via adenoviral infektion 9. Resultaterne præsenteres i dette afsnit stammer fra vores tidligere arbejde og vises her som eksempler på de særlige ke...

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

Fremgangsmåder er påvist for isoleringen af ​​sinusknude myocytter (SAMS) fra voksne mus for patch clamp elektrofysiologi eller billeddannende undersøgelser. Isolerede celler kan anvendes direkte eller kan opretholdes i kultur for at tillade ekspression af proteiner af interesse, såsom genetisk indkodede reportere.

Metoder til isolering, Kultur og funktionel karakterisering af sinoatrialt Node Myocytter 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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