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.

所有的动物程序符合由美国科罗拉多州安舒茨医学校区的大学机构动物护理和使用委员会批准的方案进行。标准协议下一直使用雄性C57BL / 6J小鼠的2-3个月的年龄进行了优化。 1.准备解决库存和用品在实验中的研究进展注：请参阅材料表进行必要的设备和用品。 <ol><li>全台氏，低钙 /镁离子台氏，卡夫改良，Brühe（KB）解决方...

<ol>
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本文提出了从成年小鼠完全分化的窦房结细胞的分离和培养的详细协议。隔离协议可靠生产适合要么直接电生理分析或随后的培养自发活动的鼠标地对空导弹。类似的协议已经报道许多其它基团（例如，参见参考文献11,12,10,13-17）。然而，我们的用于体外维持成年小鼠的SAM协议保留特性形态，自发活动，细胞的电生理特性，并允许蛋白9的腺病毒递送。 ?...

在心脏的窦房结起搏细胞（窦房结细胞“地对空导弹”）产生自发的，有节奏的动作电位（APS），通过心肌传播发起每次心跳。使用急性分离地对空导弹许多种实验已经为有助于心脏起搏器活动的产生机制，阐明至关重要。地空导弹是从他们的心房和心室肌同行中的形态，功能和表达方面有很大不同高度专业化的心肌细胞。在自组装膜自发的AP的标志是舒张驱动的膜电位向阈值，以触发下一个AP 1,2-期间自发去极化。这种“起搏器势”取决于许多不同的膜电流包括“滑稽电流”（我f）中 ，T型和L型钙电流，钠钙交换CURR的协调活动耳鼻喉科（ⅠNCX），这是由钙释放从肌质网3,4驱动。 虽然急性分离的小鼠地对空导弹是起搏的研究必不可少的实验准备，地空导弹的小鼠隔离可以通过一个具有挑战性的方法，因为鼠标SAN的模糊的解剖和小尺寸需要细致入微的显微切割和组合的酶和机械细胞解离需要仔细优化。 在这里，我们提供已被成功地用于从膜片钳记录5-8成年小鼠隔离地对空导弹的协议的详细视频演示。据我们所知，没有这样的视觉可从任何其他来源的示范。此外，一个新的方法，证明了在该分离的SAM从成年小鼠可在体外维持数天，由此允许引入蛋白质，基因编码通过腺病毒感染9报告分子或RNAi。

<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

窦房结细胞（SAMS）作为心脏的起搏器自然，倡导通过产生自发的动作电位（APS）击败每一个心脏。这些起搏器的AP反映许多膜电流和细胞内钙循环的协调活动。然而，推动自发起搏器活动地对空导弹的确切机制仍然难以实现。急性分离的地对空导弹是实验解剖心脏起搏的分子基础的必要准备。然而，模糊的解剖，显微解剖复杂和挑剔的酶消化条件阻碍了广泛使用急性分离地对空导弹。此外，方法无法获得直到最近允许地对空导弹的长期培养的蛋白表达研究。在这里，我们提供了地对空导弹从成年小鼠中分离了一步一步的协议和视频演示。方法也证明了体外和expressi维持成年小鼠地对空导弹通过对腺病毒感染外源性蛋白质。急性分离，并通过这些方法制备培养的地对空导弹是适用于各种电生理和影像学检查。

这里所描述的协议已被预先采用自发活性的SAM从成年小鼠适合于各种不同的膜片钳研究5-8的隔离。此外，该协议允许，可以在培养物中维持长达一周孤立的SAM。基因转移到培养的细胞可以通过腺病毒感染9来完成。在本节介绍的结果我们以前的工作中获得，并在这里显示为急性分离培养地对空导弹的特点例子。 ...

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

方法证实，用于从膜片钳电生理学或成像研究成年小鼠窦房结细胞（自组装膜）的分离。分离的细胞可以直接使用，也可以在培养物中保持，以允许所关注的蛋白质，如遗传编码的记者的表达。

从成年小鼠的分离，培养和窦房结肌细胞的功能分析方法

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

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

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

成人的心肌细胞的分离和遗传操作共聚焦成像

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 ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

从新生小鼠的肺血管内皮细胞的分离和培养

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

成人从人体皮肤的上皮干细胞的分离和培养

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

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

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

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

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

分离，培养和成年小鼠Cardiomyoctyes的功能分析

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 Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

从成年小鼠切牙牙源性上皮干细胞的分离和培养

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

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

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, 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 ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

成年小鼠泪液和下颌腺肌上皮细胞的分离

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...