Name: isolating primary melanocyte-like cells from the mouse heart
Uploaded: 2014-09-29T13:00:00
Description: Watch this Scientific Journal Video about Isolating Primary Melanocyte-like Cells from the Mouse Heart at JoVE.com

Dette arbejde blev støttet af National Institutes of Health award R01 HL105734 til VVPMDL understøttes delvist af National Institutes of Health Research Career Award K08 HL094748. VVP er en innovativ forsker i American Heart Association støttet af tilskud 11IRG900384.

1. Forberedelse af de nødvendige løsninger og udstyr <ol><li> Først sterilisere kirurgiske instrumenter (lige form pincet, buet form pincet, lige form saks, og buede form saks) ved autoklavering dem i 15 min. ved 121 ° C. Forbered 2 sæt instrumenter, hvor det ene sæt bruges til at dissekere ud hjerter og det andet sæt vil blive brugt til hakning op vævet. </li><li> Opløs 1 pakke MCDB153 kulturmedier i 900 ml steriliseret, filtreret vand og omrøres langsomt, indtil pulveret er opløst. Tilsæt 1,2 g natriumbi...

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	<li>Hornyak, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitf+dosage+as+a+primary+determinant+of+melanocyte+survival+after+ultraviolet+irradiation.">Mitf dosage as a primary determinant of melanocyte survival after ultraviolet irradiation.</a> Pigment Cell Melanoma Res. 22, 307-318 (2009).</li><li>Yonetani, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+expansion+of+immature+melanoblasts+and+their+ability+to+repopulate+melanocyte+stem+cells+in+the+hair+follicle.">In vitro expansion of immature melanoblasts and their ability to repopulate melanocyte stem cells in the hair follicle.</a> J. Invest. Dermatol. 128, 408-420 (2008).</li><li>Levin, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanocyte-like+cells+in+the+heart+and+pulmonary+veins+contribute+to+atrial+arrhythmia+triggers.">Melanocyte-like cells in the heart and pulmonary veins contribute to atrial arrhythmia triggers.</a> J. Clin. Invest. 119, 3420-3436 (2009).</li><li>Patel, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+insights+in+the+cellular+basis+of+atrial+fibrillation.">Novel insights in the cellular basis of atrial fibrillation.</a> Expert Rev. Cardiovasc. Ther. 8, 907-915 (2010).</li><li>Sviderskaya, E. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+neurons+and+melanocytes+induced+from+immortal+lines+of+postnatal+neural+crest-like+stem+cells.">Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells.</a> FASEB J. 23, 3179-3192 (2009).</li><li>Sviderskaya, E. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P16Ink4a+in+melanocyte+senescence+and+differentiation.">P16Ink4a in melanocyte senescence and differentiation.</a> J. Natl. Cancer. Inst. 94, 446-454 (2002).</li><li>Cook, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+melanoblasts+in+culture:+Expression+of+BRN2+and+synergistic+regulation+by+fibroblast+growth+factor-2,+stem+cell+factor,+and+endothelin-3.">Human melanoblasts in culture: Expression of BRN2 and synergistic regulation by fibroblast growth factor-2, stem cell factor, and endothelin-3.</a> J. Invest. Dermatol. 121, 1150-1159 (2002).</li><li>Fang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+conditions+for+the+generation+of+melanocytes+from+human+embryonic+stem+cells.">Defining the conditions for the generation of melanocytes from human embryonic stem cells.</a> Stem Cells. 24, 1668-1677 (2006).</li><li>Brito, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timeline+and+distribution+of+melanocyte+precursors+in+the+mouse+heart.">Timeline and distribution of melanocyte precursors in the mouse heart.</a> Pigment Cell Melanoma Res. 21, 464-470 (2008).</li><li>Yajima, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+of+heart+melanocytes+is+specified+and+the+level+of+pigmentation+in+the+heart+may+correlate+with+coat+color.">The isolation of heart melanocytes is specified and the level of pigmentation in the heart may correlate with coat color.</a> Pigment Cell Melanoma Res. 21, 471-476 (2008).</li></ol>

Hjerte melanocytter er til stede i anatomiske steder i hjertet, der almindeligvis giver anledning til atriearytmien udløsere. Disse regioner omfatter lungevenerne, atrioventrikulær ringformede, bageste venstre forkammer og foramen ovale (figur 4). For at forstå den rolle, disse celler spiller i arrhythmogenesis udvikler vi teknikker til at isolere dem og studere deres fysiologi. Vi erkendte dog, at forbedring af denne teknik til at give sundere og større mængder af kardiologiske melanocytter ville ...

Vi har for nylig identificeret en ny celle population i hjertet af mus og mennesker, der udtrykker melanin syntese enzymer og morfologisk ligner kutane melanocytter. Vi kaldes disse cellers kardiale melanocytter &#39;og fundet de er til stede i anatomiske steder, hvorfra atriearytmien udløser ofte oprindelse i mennesker. Vi fandt også kardiologiske melanocytter bidrage til atrial arytmi i mus med germlinie sletning af DCT. Hjerte melanocytter er tyndt fordelt i hele musen atrium, hvor de omfatter mindre end 0,1% af alle atrielle celler. For at undersøge de elektriske og biologiske egenskaber kardiologiske melanocytter vi udviklet en procedure for at isolere dem fra muse-hjerte ved hjælp af Cre-inducerbare melanocytstimulerende specifikke markører. Vores indledende procedure blev udviklet fra dem, der anvendes til at isolere neonatale murine cardiomyocytter og gav meget lille antal celler, der er egnede til patch clamp optagelser eller PCR-analyse. For at forbedre helbredeth og levedygtighed af hjerte melanocytter, for mere dybtgående elektrofysiologiske analyser eller biokemiske undersøgelser, har vi udviklet en raffineret procedure fra dem, designet til at isolere kutane melanocytter. Den beskrevne og demonstreret i denne anmeldelse fremgangsmåde har den fordel at producere sundere kardielle melanocytter, der kan være belagt som en ren population eller med kardiomyocytter.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>MCDB 153</td>
 <td>Sigma-Aldrich</td>
 <td>M7403</td>
 <td>Dissolve in: Water</td>
 </tr>
 <tr>
 <td>DPBS, no calcium no magnesium</td>
 <td>Invitrogen</td>
 <td>14190136 </td>
 <td>Stock Conc.: 1X 
 Working Conc.: 1X</td>
 </tr>
 <tr>
 <td>Sodium Bicarbonate, or Sodium Bicarbonate Solution, 7.5% </td>
 <td>Sigma-Aldrich </td>
 <td>S5761 or S8761 </td>
 <td>Water 
 Stock Conc.: 1X 
 Working Conc.: 1X</td>
 </tr>
 <tr>
 <td>Penicillin-streptomycin </td>
 <td>Invitrogen</td>
 <td>15140163 </td>
 <td>Stock Conc.: 100X 
 Working Conc.: 1X</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum</td>
 <td>Life Technology</td>
 <td>Certified</td>
 <td>Working Conc.: 4%</td>
 </tr>
 <tr>
 <td>TPA</td>
 <td>Sigma-Aldrich</td>
 <td>P1585</td>
 <td>DMSO 
 Stock Conc.: 3.2 &mu;M 
 Working Conc.: 32 nM</td>
 </tr>
 <tr>
 <td>Transferrin</td>
 <td>Sigma-Aldrich</td>
 <td>T8158</td>
 <td>DPBS 
 Stock Conc.: 0.1 mg/ml 
 Working Conc.: 1 &mu;g/ml </td>
 </tr>
 <tr>
 <td>Dibutyryl cyclic AMP</td>
 <td>Sigma-Aldrich</td>
 <td>D0627</td>
 <td>DPBS 
 Stock Conc.: 0.5 mM 
 Working Conc.: 5 &mu;M</td>
 </tr>
 <tr>
 <td>Insulin</td>
 <td>Sigma-Aldrich</td>
 <td>I6634</td>
 <td>10 mM 
 acetic acid 
 Stock Conc.: 1 mg/ml 
 Working Conc.: 5 &mu;g/ml</td>
 </tr>
 <tr>
 <td>Basic FGF</td>
 <td>BD Biosciences</td>
 <td>354060</td>
 <td>Water 
 Stock Conc.: 4 ng/&mu;l 
 Working Conc.: 4 ng/ml</td>
 </tr>
 </tbody>
</table>

isolating primary melanocyte-like cells from the mouse heart

Vi identificerede en ny population af melanocytstimulerende lignende celler (også kendt som kardiale melanocytter) i hjerterne på mus og mennesker, der bidrager til atriearytmien triggers i mus. For at undersøge de elektriske og biologiske egenskaber kardiologiske melanocytter vi udviklet en procedure for at isolere dem fra mus hjerter, vi stammer fra dem, designet til at isolere neonatale murine kardiomyocytter. For at opnå sundere kardiale melanocytter er egnede til mere omfattende patch clamp eller biokemiske undersøgelser, har vi udviklet en raffineret fremgangsmåde til isolering og forkromning kardiale melanocytter baseret på dem, der oprindeligt designet til at isolere kutane melanocytter. Den raffinerede procedure påvises i denne anmeldelse og producerer større mængder af sunde melanocytstimulerende-lignende celler, som kan være belagt som en ren population eller med kardiomyocytter.

Beskrevet i denne artikel protokol er en forbedret teknik i forhold til den, vi tidligere offentliggjorte til isolering kardiale melanocytter fra den murine hjerte. Denne procedure anvender brugen af ​​fluorescerende markører drevet af melanin syntese enzym-specifik Cre-rekombinase at mærke kardiale melanocyt-lignende celler. Det er vigtigt at bruge en markør til at mærke kardiale melanocytter, når der arbejdes med frisk isolerede embryoner eller neonatale celler, idet de morfologiske forskelle, der adskiller k...

Watch this Scientific Journal Video about Isolating Primary Melanocyte-like Cells from the Mouse Heart at JoVE.com

Isolating Primary Melanocyte-like Cells from the Mouse Heart

I denne protokol, w e identificeret en ny population af melanocytstimulerende lignende celler (også kendt som kardiale melanocytter) i hjerterne på mus og mennesker, der bidrager til atriearytmien udløser i mus.

Isolering af primær melanocytstimulerende lignende celler fra mus Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

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