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

这项工作是由卫生部授予R01 HL105734到VVPMDL国家研究院​​赞助支持，部分是由健康研究事业奖K08 HL094748国家机构。 VVP是美国心脏协会的创新研究员被授予11IRG900384支持。

1，准备必要的解决方案和设备<ol><li>首先，通过高压灭菌他们15分钟消毒手术器械（直形镊子，弯形镊子，直板造型剪刀和弯曲的形状剪）。在121℃。准备2台仪器，其中一组用于解剖出来的心和另一组中的将被用于切碎起来的组织。 </li><li>溶解MCDB153培养基1包900毫升的消毒，过滤后的水，搅拌缓慢，直到粉末完全溶解。添加1.2克碳酸氢钠，或毫升钠15.7碳酸氢盐溶液，对于每升培养基的最终体积...

<ol>
	<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>

心黑素细胞存在于心脏内的解剖位置中通常引起心律失常的触发器。这些区域包括肺静脉，房室瓣环，后左心房和卵圆孔（ 图4）。要理解中的作用，这些细胞在心律失常玩，我们开发的技术来隔离他们，研究他们的生理机能。但是，我们认识到，提高该技术以产生更健康，更大量的心脏黑色素细胞将允许进一​​步的研究，以探讨心脏黑色素细胞对外源性刺激的反应。此外，改进的...

我们最近发现在表达黑色素合成酶和形态上类似皮肤的黑素细胞的小鼠和人类的心脏了一种新的细胞群。我们称这些细胞的黑素心“，发现他们是存在于解剖位置，从该房性心律失常的触发往往起源于人类。我们还发现心脏黑色素细胞在小鼠生殖细胞缺失的DCT导致房性心律失常。心黑素细胞稀疏地分布在整个小鼠心房，他们包括所有心房细胞少于0.1％。调查心脏黑色素细胞的电特性和生物特性，我们开发了一种方法，从使用Cre重组酶诱导的黑素细胞特异性标记物的小鼠心脏隔离。我们的初始步骤，从那些用于分离新生鼠心肌细胞，并产生非常小的数值适用于膜片钳记录或PCR分析细胞的开发。然而，为了提高愈合日和心脏的黑色素细胞，进行更深入的分析，电生理或生化研究的可行性，我们制定了从那些旨在隔离皮肤的黑素细胞精致的过程。描述和展示了在本次审查的程序有生产，可作为镀纯人群或心肌与心脏健康的黑素细胞的优势。

<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

我们确定了一个新的黑色素细胞样细胞群（也称为心源性黑色素细胞），在小鼠中导致房性心律失常触发小鼠和人类的心。调查心脏黑色素细胞的电特性和生物特性，我们开发了一种程序，从小鼠心脏，我们从这些设计以分离新生鼠心肌细胞衍生的隔离。为了获得适合于更广泛的膜片钳或生化研究健康心脏的黑素细胞，我们开发了一种精制方法分离和镀基于那些最初设计以隔离皮肤黑素细胞中黑素细胞的心脏。精致的过程证明了本次审查，并产生更大的健康黑色素细胞样细胞，可作为镀纯人口或心肌细胞的数量。

在这篇文章中描述的协议是相对于我们以前出版的从小鼠心脏心脏分离黑素细胞之一的改进技术。这个过程采用了利用由黑色素合成酶特异性Cre重组酶从动标注心脏黑色素细胞样细胞的荧光标记物。使用的标记物与新鲜分离的胚胎或新生儿细胞中工作时，由于该区分心脏的黑素细胞从心房肌细胞的形态差异尚未发展（ 图1A）标记心脏的黑素细胞是重要的。 如图1A时，分离...

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

在这个协议中，W E 发现了新的黑色素细胞样细胞群（也称为心源性黑色素细胞）的小鼠和人类中，有助于房性心律失常引发小鼠的心。

从小鼠心脏中分离主黑素细胞样细胞

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 ...

Research

JoVE Journal

Biology

Primary Culture of Adult Rat Heart Myocytes

成年大鼠心脏心肌细胞的原代培养

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

科研

生物学

Isolating Stem Cells from Soft Musculoskeletal Tissues

从软肌肉骨骼组织中分离干细胞

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

Clonogenic Assay: Adherent Cells

克隆形成含量：贴壁细胞

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

从细胞中一氧化氮和超氧负离子自由基的电子顺磁共振光谱的检测，使用自旋陷阱

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

选择和隔离殖民地的人类诱导多能干细胞重新编程成年成纤维细胞

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

从发展胚胎小鼠心脏瓣膜区蛋白分离

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

小鼠视网膜色素上皮细胞的原代培养

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

成人小鼠心脏干细胞的分离、鉴定及分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...