从成年小鼠的分离，培养和窦房结肌细胞的功能分析方法
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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。
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Divulgations
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Sylgruard/Elastomer Kit | Dow Corning | 184 SIL ELAST KIT 0.5KG | |
| Borosilicate 9" pasteur pipettes | Fisher Scientific | 13-678-20C | |
| Small, round bottomed culture tubes | Fisher Scientific | 352059 | |
| Large, round bottomed culture tubes | Corning | 14-959-11B | |
| Elastase | Worthington Biochemical | LS002279 | |
| Liberase TM | Roche | 5401119001 | Tissue dissociation solution |
| Heparin | SAGENT Pharmaceuticals | NDC 25021-400-10 | |
| Mouse Laminin | Corning | CB-354232 | |
| 12 mm round glass coverslips | Fisher | 12-545-80 | |
| 24-well culture plate | Fisher | 08-772-1 | |
| Ad-mCherry | Vector Biolabs | 1767 | |
| Ad-eGFP | Vector Biolabs | 1060 | |
| Plastic, disposable transfer pipette | Fisher Scientific | ||
| Micro scissors | Fisher Scientific | 17-467-496 | |
| Dumont #4 Forceps | Roboz Instruments | RS-4904 | |
| Tissue Forceps | Roboz Instruments | RS-8164 | |
| Dissecting Iris Scissors | WPI, Inc. | 501264 | |
| Dissecting Pins | Fine Science Tools | 26002-20 | |
| NaCl | Sigma | 71376 | |
| KCl | Sigma | 60128 | |
| KH2PO4 | Sigma | 60353 | |
| HEPES | Sigma | 54457 | |
| glucose | Sigma | G0350500 | |
| MgCl2 | Sigma | M8266 | |
| CaCl2 | Sigma | C1016 | |
| taurine | Sigma | T0625 | |
| BSA | Sigma | A2153 | |
| K-glutamate | Sigma | G1501 | |
| K-aspartate | Sigma | A6558 | |
| MgSO4 | Sigma | M7506 | |
| creatine | Sigma | C0780 | |
| EGTA | Sigma | E3889 | |
| Mg-ATP | Sigma | A9187 | |
| Amphotericin-B | Fisher Scientific | 1397-89-3 | |
| Isoproterenol | Calbiochem | 420355 | |
| Media199 | Sigma | M4530 | |
| 2,3-butanedione monoxime (BDM) | Sigma | B0753 | |
| Fetal Bovine Serum (FBS) | Sigma | SH30071 | |
| Bovine Serum Albumin (BSA) | Sigma | A5611 | |
| Insulin | Sigma | I3146 | |
| Transferrin | Sigma | I3146 | |
| Selenium | Sigma | I3146 | |
| Penicillin | GE Healthcare | SV30010 | |
| Streptomycin | Hyclone | SV30010 | |
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