部分一，准备进行手术<ol><li>大鼠手术的前一天，确信所有的解决方案，但不添加任何酶。缓冲器A，B和洗涤缓冲液都应该被过滤为不育之前存储。缓冲区都应该保存在4 ° C </li><li> 10-20微克/毫升层粘连蛋白在2 mL 1XSFM以及前一天必须镀有6孔组织培养板，并应存放在37 ° C中的CO 2培养箱。 </li><li>在手术当天，准备用70％乙醇消毒钳，剪刀，镊子，手术器械，并让他们干纸巾。 </li><li>充满0.5 ml肝素溶液（90 U / ml）的1 mL注射器准备。 </li><li>现在是很好的做法，灌注设备的运行，通过70％的乙醇，使用逆蠕动泵洗。完成后用乙醇冲洗，用双蒸水冲洗设备，最后再冲洗干净用缓冲液A的流量，通过在37℃水浴中放置了一个烧杯收集。 </li><li>要准备清洗的灌注设备，填补40毫升缓冲液A和左边的注射器管40毫升的缓冲B.正确的注射器管</li><li>总理灌注管的导管，使缓冲液A流经仪器提示。填充到缓冲区的40毫升的注射器。确保没有空气气泡被困在管这一步后。 </li><li>现在，关闭缓冲区注射器活塞阀，并用缓冲液B等重复的过程，灌注管现在准备用缓冲液B。 </li><li>最后，灌注设备是用缓冲液B活塞阀打开，但流停止使用稳压器，四线。 </li><li>丢弃在收集烧杯的缓冲流虽然。 </li><li>手术前做的最后一件事情是添加所需的酶原液，然后过滤酶液，就像其他的解决方案筛选的前一天。过滤后，该解决方案可以在室温下。 </li></ol>第二部分。大鼠心脏解剖<ol><li>下列标准协议安乐死与异氟醚对大鼠在毒气室。 </li><li>消毒用70％乙醇的胸部切口的面积。 </li><li>用剪刀开胸，暴露心脏。 </li><li>注入0.5 ml肝素溶液（90 U / mL）的左心室。 </li><li>取出心脏主动脉完好无损，地方大部份在冰冷的缓冲B. </li></ol>第三部分。心肌细胞隔离<ol><li>如果下面的过程是正确地坚持一个典型的大鼠心脏应该产生一个杆状的心肌细胞的高分数（而不是死四舍五入细胞）。 </li><li>首先，钳主动脉导管。推入心导管尖端不应太远，以确保通过冠状动脉灌注对心脏有好处。 </li><li>一旦夹住，领带主动脉导管一期缝合。 </li><li>接下来，打开四线调节器，允许快速滴速（〜60滴/ min），并允许缓冲液B洗心。 </li><li>在缓冲液B洗，用小剪刀和镊子去除心房和执着心的任何脂肪或肺组织。 </li><li>缓冲液B完成后，切换流缓冲液A </li><li>虽然缓冲区，一个是允许流量为5分钟，几个小任务，应迅速执行。 </li><li>首先，温暖的酶液在37℃水浴。 </li><li>最后，当一个缓冲区是从注射器（但目前仍然在管）耗尽，负载在注射器50毫升酶液的灌注设备</li><li>现在有必要放弃所有的流量通过从收集烧杯中，水浴（吹打），直到酶液perfuses心。 </li><li>只要酶液perfuses心，激活设置从收集烧杯转移酶液，以补充注射器答：蠕动泵</li><li>允许的酶液，通过心脏的流量为10分钟。心消化，它开始显得臃肿。 </li><li>添加37.5μL0.1 M的氯化钙2酶液在注射器一个有效浓度为0.1毫米的Ca 2 +。 </li><li> 10分钟后，钙离子浓度增加至0.2毫米，通过增加一个额外的50μL0.1 M的氯化钙 2注射器，让灌注为10分钟出发。 </li><li>切断心室，并转移到一个小的无菌烧杯中含有20毫升酶液。 </li><li>在烧杯中的钙离子浓度增加至0.4毫米，加入40μL0.1 M的氯化钙 2 。 </li><li>轻轻剁碎成10个或更多件的心，一双小无菌剪刀。 </li><li> 5分钟的烧杯在37℃，温和的摇摆。 </li><li>加入40毫升0.1 M的氯化钙2有效浓度为0.6毫米的Ca 2 +和心片轻轻磨碎，用塑料转移吸管3-5倍。 </li><li>一个额外的5分钟的孵化后，在37 ° C，加入40毫升0.1 M 的氯化钙2，轻轻磨碎像以前那样。 </li><li>单独的消化，从单一的心肌细胞非消化结缔组织，通过无菌500微米网使用的过滤。 </li><li>允许细胞在室温下10分钟在50毫升管定居。 </li><li>倒掉上清转移吸管。 </li><li>轻轻重悬在洗涤缓冲液＃1中的细胞，让细胞在室温为10-20分钟解决。 </li><li>虽然细胞沉淀，取一小等份，并使用这个时候，作为一个机会来评估一个倒置显微镜下，悬浮液中的细胞的质量和可行性。一个很好的准备，将有一个高杆像清脆的条纹细胞的比例（&gt; 80％）。 </li><li>一旦细胞相继落户，弃上清，轻轻悬浮细胞洗涤缓冲液＃2。让细胞在室温下解决，这应该再次需要10-20分钟，并弃去上清液。 </li><li>再次重复洗涤过程中洗涤缓冲液＃3，细胞将文化准备。 </li></ol>第四部分。心肌细胞培养<ol><li>轻轻重悬细胞在5％的血清媒体。在转让细胞组织培养板之前，用1X可持续森林管理的板块。转移细胞所需的密度和中的CO 2组织培养孵化器的孵化，为3-5小时。 </li><li>媒体切换到1X可持续森林管理。 </li><li>细胞可培养四天，用来作为实验的要求。 </li></ol>第五部分代表性的结果/成果应该有70％以上，在倒置显微镜下活的心时，该协议是正确的心肌细胞。 <img src="/files/ftp_upload/1308/fig1.jpg" alt="图1" /> 表1：溶液配方为1升10倍的Ca 2 +无KH缓冲液 ： <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td>质量</td><td>终浓度</td></tr><tr><td>氯化钠</td><td> 68.96摹</td><td> 1180 mM的</td></tr><tr><td>氯化钾</td><td> 3.58摹</td><td> 48毫米</td></tr><tr><td> HEPES </td><td> 59.58摹</td><td> 250毫米</td></tr><tr><td> K 2 HPO 4 </td><td> 2.85摹</td><td> 12.5毫米</td></tr><tr><td> 硫酸镁 </td><td> 3.08摹</td><td> 12.5毫米</td></tr></tbody></table> 表2：1升缓冲液A的溶液配方 ： 添加1.98克葡萄糖100毫升10X锦缓冲区，并携带一个1升的最后体积与葡萄糖的渗透压调节到接近生理条件下（〜300）。带来的pH值用NaOH 7.4。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> - </td><td>地下</td><td>终浓度</td></tr><tr><td> 10X KH缓冲</td><td> 100毫升</td><td> 100毫升</td></tr><tr><td>血糖</td><td> 1.98摹</td><td> 11毫米</td></tr><tr><td>置于1升</td><td></td><td></td></tr><tr><td>置于pH值用NaOH 7.4 </td><td></td><td></td></tr><tr><td colspan="3">调整渗透压接近生理条件下与葡萄糖（〜300）。 </td></tr></tbody></table> 表3：酶缓冲液的解决方案处方<table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td>质量</td><td>终浓度</td></tr><tr><td>二型胶原酶</td><td> 25毫克</td><td> 0.05％（W / V） </td></tr><tr><td>蛋白酶第十四</td><td> 10毫克</td><td> 0.02％（W ​​/ V） </td></tr><tr><td> BDM </td><td> 0.025摹</td><td> 5毫米</td></tr><tr><td>肉碱</td><td> 0.020摹</td><td> 2毫米</td></tr><tr><td>牛磺酸</td><td> 0.031摹</td><td> 5毫米</td></tr><tr><td>谷氨酸</td><td> 0.020摹</td><td> 2毫米</td></tr><tr><td> 0.1 M的氯化钙2 </td><td> 12.5微升</td><td> 25 UM </td></tr><tr><td>缓冲液A </td><td></td><td>填充至50 ml </td></tr></tbody></table> 表四：解决方案处方25X BDM /牛磺酸/ BSA（B / T / B） <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td>质量</td><td>终浓度</td></tr><tr><td> BDM </td><td> 0.076摹</td><td> 125毫米</td></tr><tr><td>牛磺酸</td><td> 0.094摹</td><td> 125毫米</td></tr><tr><td>牛血清白蛋白</td><td></td><td> 0.1％（W / V） </td></tr><tr><td>缓冲液A </td><td></td><td>填充至6 ml </td></tr></tbody></table> 表5：缓冲区B和清洗缓冲的解决方案处方<table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 缓冲液： </td><td> 乙 </td><td> ＃1 </td><td> ＃2 </td><td> ＃3 </td></tr><tr><td> 地址： </td><td></td><td></td><td></td><td></td></tr><tr><td> 0.5 M的氯化钙2 </td><td> 0.4毫升</td><td> 0.1毫升</td><td> 0.125毫升</td><td> 0.15毫升</td></tr><tr><td> 氯化钙终浓度为2 </td><td> 1毫米</td><td> 1毫米</td><td> 1.25毫米</td><td> 1.5毫米</td></tr><tr><td> 25X B / T / B </td><td> --- </td><td> 2毫升</td><td> 2毫升</td><td> 2毫升</td></tr><tr><td>缓冲到最终卷</td><td> 200毫升</td><td> 50毫升</td><td> 50毫升</td><td> 50毫升</td></tr></tbody></table> 表6：培养媒体的解决方案处方<table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 2X无血清培养基（SFM）， </td><td></td><td></td></tr><tr><td rowspan="2"> </td><td rowspan="2"></td><td> 终浓度 </td></tr><tr><td> （稀释后） </td></tr><tr><td>肉碱</td><td> 0.099摹</td><td> 10毫米（5毫米） </td></tr><tr><td>牛磺酸</td><td> 0.063摹</td><td> 10毫米（5毫米） </td></tr><tr><td>肌酸</td><td> 0.075摹</td><td> 10毫米（5毫米） </td></tr><tr><td>抗生素/ Antimycotic </td><td> 0.5毫升</td><td> 1％（0.5％）（V / V） </td></tr><tr><td>媒体199 </td><td> 45毫升</td><td>填充至50 mL </td></tr><tr><td></td><td></td><td></td></tr><tr><td> 1X可持续森林管理 </td><td></td><td></td></tr><tr><td></td><td></td><td> 终浓度 </td></tr><tr><td> 2X可持续森林管理</td><td> 25毫升</td><td> 1X </td></tr><tr><td>媒体199 </td><td></td><td>填充至50 mL </td></tr><tr><td></td><td></td><td></td></tr><tr><td> 1X血清媒体5％ </td><td></td><td></td></tr><tr><td></td><td></td><td> 终浓度 </td></tr><tr><td> 2X可持续森林管理</td><td> 25毫升</td><td> 1X </td></tr><tr><td>胎牛血清</td><td> 2.5毫升</td><td> 5％（V / V） </td></tr><tr><td>媒体199 </td><td></td><td>填充至50 mL </td></tr></tbody></table>

<ol>
	<li>Miriyala, J., Nguyen, T., Yue, D. T., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+CaVbeta+subunits,+and+lack+of+functional+reserve,+in+protein+kinase+A+modulation+of+cardiac+CaV1.2+channels.">Role of CaVbeta subunits, and lack of functional reserve, in protein kinase A modulation of cardiac CaV1.2 channels.</a> Circ Res. 102 (7), e54-e64 (2008).</li><li>SX, T. a. k. a. h. a. s. h. i., Mittman, S., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinctive+modulatory+effects+of+five+human+auxiliary+beta2+subunit+splice+variants+on+L-type+calcium+channel+gating.">Distinctive modulatory effects of five human auxiliary beta2 subunit splice variants on L-type calcium channel gating.</a> Biophys. , 84-845 (2003).</li></ol>

关键的一步，这是挂在离体心脏灌注系统的速度。酶消化期的长短可能会有点不同的各组大鼠。调整取决于软心定期的消化期后成为。 “缓慢复苏的Ca 2 +内切酶消化后获得的Ca 2 +宽容健康的细胞至关重要。 豚鼠，该协议是相同的，除了透明质酸是第十四蛋白酶，而不是使用。通常情况下，我们发现，最初的活细胞百分比是豚鼠至大鼠相比，虽然他们生存，仅...

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		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S6191</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P9541</td>
<td>&nbsp;</td>
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<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H4034</td>
<td>&nbsp;</td>
<tr>
<td>K2HPO4</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P9666</td>
<td>&nbsp;</td>
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<td>MgSO4</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>63068</td>
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<td>&nbsp;</td>
<td>Sigma</td>
<td>G7021</td>
<td>&nbsp;</td>
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<td>CaCl2</td>
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<td>Sigma</td>
<td>C7902</td>
<td>&nbsp;</td>
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<td>Heparin Sodium Salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H4784</td>
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<td>Worthington Biochemical</td>
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<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Sigma</td>
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<td>&nbsp;</td>
<td>Sigma</td>
<td>C9500</td>
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<td>Sigma</td>
<td>T0625</td>
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<td>&nbsp;</td>
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<td>354232</td>
<td>&nbsp;</td>
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<td>water bath</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>15-462-5</td>
<td>&nbsp;</td>
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<td>variable flow chemical pump</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>15-077-67</td>
<td>&nbsp;</td>
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<td>6-well culture plate</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3516</td>
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primary culture of adult rat heart myocytes

原代培养成年啮齿类动物的心脏细胞，心血管研究的一个重要的模型系统。不过，建立强大的，可行的的培养成人心肌细胞可以在技术上具有挑战性的，速率限制，许多研究者的一步。在这里，我们描述了一个协议，以获得一个可行的几天停留在文化的成年大鼠心脏心肌细胞的高产。心是孤立的，与胶原酶和蛋白酶灌注下低钙<sup&gt; 2 +</sup&gt;恢复单个心肌细胞的条件。钙<sup&gt; 2 +</sup&gt;容错细胞得到逐步增加细胞外Ca<sup&gt; 2 +</sup&gt;集中在三个随后的清洗步骤。被过滤，在培养液中悬浮细胞，并镀金层粘连蛋白涂单。使用此协议培养的心肌细胞获得长达4天的可行和适合最，包括电生理学，生物化学，影像和分子生物学的实验。

Watch this Scientific Journal Video about Primary Culture of Adult Rat Heart Myocytes at JoVE.com

Primary Culture of Adult Rat Heart Myocytes

在本文中，我们描述了一个典型的成年大鼠心脏心肌细胞分离和培养。胶原酶和蛋白酶消化，分离单个心肌细胞。心肌细胞培养遵守此协议满足大多数实验要求。

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 ...

primary-culture-of-adult-rat-heart-myocytes

Research

JoVE Journal

Biology

20.2K 次观看.  Columbia University. 在本文中，我们描述了一个典型的成年大鼠心脏心肌细胞分离和培养。胶原酶和蛋白酶消化，分离单个心肌细胞。心肌细胞培养遵守此协议满足大多数实验要求。

视频: Primary Culture of Adult Rat Heart Myocytes

Organotypic Culture of Adult Rabbit Retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with imaging techniques, genetic manipulation, and electrophysiological recording. Here we present a simple interphase tissue culture system for adult rabbit retina that requires no specialized equipment and very little maintenance. We demonstrate the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Rabbit retinas cultured this way can be kept alive for up to 6 days with very little changes of the overall anatomical structure or the morphology of individual ganglion- and amacrine cells.

This article demonstrates the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells.

器官型培养的成年兔视网膜

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

科研

生物学

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

大鼠心脏中植入的工程组织

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

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 a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

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 ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

建立从小学成人成纤维细胞培养鼠害

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

高度耦合的大鼠心肌线粒体的制备

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

大鼠肝细胞的分离和小学文化

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

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). ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

从成年大鼠心脏同时分离高质量心肌细胞，内皮细胞和成纤维细胞

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

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 defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

来自新生儿和成年鼠皮肤的原代小鼠角化细胞的分离和培养

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 ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

大鼠肾上腺皮质细胞的原代培养及非甾体功能的测定

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...