The Watts Laboratory through the years, and Dr. Marlene Cohen and Kathy Schenck for teaching us this assay over two decades ago. NIGMS R25GM074119 supported development of this teaching module for a Short Course in Integrative and Systems Pharmacology held on the campus of MSU from 2005 to 2013.

注：本文中所描述的所有程序都是根据密歇根州立大学的机构动物护理和使用委员会（IACUC）制定的准则进行。 1.系统准备和设置<ol><li>使5升的生理盐溶液（PSS），这是需要的，它使用高达50毫升组织浴的组织浴中的收缩实验的量; 见表2 PSS配方。乘以组织浴的数目乘以浴体积，然后通过所需的组织洗涤次数乘以计算总所需体积。 <ol><li>使用表2为使PSS的指南。盐溶解在约4升的水。注：HPLC - I类水推荐</li><li> （如果使用的二水合物盐147克/ L）的溶液中，所以最终的解决方案是1.6毫钙加入8毫升的1M的CaCl 2溶液。 </li><li>量子sufficit PSS解决5 L. </li></OL&gt; </li><li>通过打开循环恒温水浴预热组织浴系统至37℃。关键的步骤：在系统的每一个组件是水夹套，确保它们串联连接到彼此。流的方向是至关重要 - 确保在最高有倒钩连接的水流入各组分在最低的有倒钩的连接和缩小。 </li><li>开启数据采集系统。电源上的力传感器至少15分钟前实验平衡温度。 注：大多数力传感器采用应变计是温度变化敏感，表现出热漂移最初通电后。 </li><li>启动数据采集软件，并确保与数据采集系统连接。请按照制造指令使数据记录。 </li><li>确保力传感器组织放置在组织浴和数据记录开始之前，前被校准; FOllow制造商的说明进行校准。 </li><li>组织浴系统连接到一个95％ 的 O 2/5％CO 2的医用级气筒和检查气体泄漏，然后加压系统。 </li><li>填充组织浴储层PSS和允许溶液的时间来达到最佳温度。首相系统并删除系统和管道内的气泡。 </li><li>检查组织浴充气器，以确保一致溶液曝气，其中增氧PSS缓冲，并提供布朗运动来分发，这将在组织浴中的试验过程中被引入药物。确保通风/气泡不会引起组织运动，这将扰乱数据记录;降低气体流量是必要的。 注：该组织浴和数据采集系统现在已经准备好开始实验。 </li></ol> 2.组织准备<ol><li>理想地，解剖来自动物组织之前立即使用，并直接将我n要PSS。 注意：有些组织可以在一夜之间在4℃保存在PSS，但适当的控制必须做到以验证长期存放组织功能;见第6。 </li><li>使用胸主动脉作为这项工作的组织。 </li><li>麻醉按照机构准则大鼠和向下放置鼠背侧。通过脚趾捏并为此疼痛刺激反射反应的损失确认麻醉。 注：此协议使用70毫克/公斤的戊巴比妥通过腹腔注射交付。对啮齿动物的麻醉制度的指导方针可能会有所不同。 </li><li>通过创建一个切口沿隔膜在肋条笼形件的底部剪刀创建气胸。然后，继续从胸腔的底部切口的胸骨和平分。 </li><li>解剖远或放置一旁那些器官是在脊柱的顶部和定位在主动脉，其直接位于沿脊柱。 注意：这个STEP提供了明确的工作空间解剖出主动脉。 </li><li>断绝一切联系的主动脉;从食道，肺等，并切断主动脉垂直于脊柱在膜片的水平。 </li><li>轻轻握住钳主动脉，用剪刀从脊柱解剖主动脉。开始在邻近的脊椎和工作朝向心脏的下隔膜面积的夹层。请务必采取额外的预防措施不拉或拽主动脉组织，这可能会损坏组织。 </li><li>立即将含有PSS的准备解剖盘的主动脉。 注：主动脉成环的解剖是在解剖盘，有一个黑色的硅橡胶基础最好的做法。这允许使用的电线可用于导管插入主动脉和固定到盘的，从而提供稳定性，同时在解剖盘中。黑色背景对比提供了有助于解剖。应注意在使用cannulating线为endothel胶质细胞层可以用与容器的内腔的导线的多余的摩擦被除去。 </li><li>取的线和一个小的90°角，另一端成角度的端部推入硅橡胶基础锚定导丝。放置在解剖盘整个主动脉。 </li><li>持导丝与镊子的末端，然后将金属丝轻轻拧到主动脉的内腔。 </li><li>使用镊子和主动脉滑到线。当导丝的自由端是可见的，曲线的丝轻轻并将自由端插入皿的硅橡胶基础，以稳定的组织。 </li><li>使用小vannas剪刀和镊子，取出血管周围脂肪组织和所有其他外部组织，血块等直到胸主动脉出现白色和略有纤维性。 </li><li>从电线拆下清洗主动脉，用剪刀剪下的戒指是大约3-5毫米宽的主动脉。 </li><li>放置一个主动脉环上的一对钛中的一个ssue钩握住钩子和使用镊子轻轻滑动环到挂钩。与第二钩状重复此过程。请注意，以确保挂钩不纠结;参见图3。 </li><li>检查每个钩具有连接不同的丝线。检查一个钩丝具有允许用于连接到一个玻璃或不锈钢棒的小打结循环，而另一种是10-4厘米长片缝合，就可以用手绑在力传感器;参见图3。 注意：一旦主动脉固定在钩，所述组织是准备被安装在组织浴;参见图3。 </li><li>重复步骤2.14-2.15，安装第二主动脉环在第二组织浴室。 </li></ol>巴斯3.组织安置<ol><li>注意，在这个系统中，组织浴本身是固定的。此时，填充温热，充气的PSS的组织浴中，并允许该溶液来。温度即</li><li>用丝线缝合，打结钩在组织上，以制备在不锈钢棒上的塞销中的一个。将为此进入组织浴室。杆连接到环立场，并将在染色菜充满了PSS，以保持组织沉浸在缓冲区中的另一端;参见图3。 <ol><li>放置杆和组织在组织浴室，并确保组织被完全沉浸在PSS和杆是安全的;参见图3。 </li><li>配合其他缝合力传感器，并确保离开松弛的组织和力传感器之间的缝合。 注：此松弛会通过调整微米被删除;参见图3。 </li></ol></li><li>重复这些步骤，第二主动脉瓣环和组织洗浴室。 </li></ol> 4.设置被动张力注意：每个组织具有长度（螺），在该平滑肌细胞响应最优LLY。初步实验，以确定最佳的拉伸张力而实现待检查该长度必须为每个单独的组织类型进行。大鼠胸主动脉有4个g的最优被动伸展紧张。 <ol><li>使用千分尺/齿条和小齿轮，以增加张力至2g，并等待所述组织，以达到平台期。一旦达到平稳状态，增加紧张又2克，等待组织高原。 注：在主动脉，后2克的初始张力树立和平台达到，组织应再放宽到〜1.6克2克的第二应用将使组织到〜3.6克，从该组织将再次放松和张力将减小。因此，具有加入4- g总张力之后，该软件应说明大致3.2克张力。 </li><li>重复这些步骤，第二主动脉瓣环和组织洗浴室。 </li></ol> 5.平衡和药品制作<ol><li>平衡组织的施加被动张力至组织后的60分钟。 </li><li>在平衡期，洗组织每次15-20分钟。沥干组织浴缸和一个温暖水库替换PSS的PSS。 注意：在此期间，该组织将放松，这是由被动张力的损失表示。 </li><li>在此期间，准备药物用于实验，这需要至少1000倍，在浴中的实际浓度越浓，所以只有小体积药物库存的需要以达到所需的浓度。 </li></ol> 6.初次挑战<ol><li>在平衡期结束时，洗组织最后一次，保存数据，并重新零的所有输入。 </li><li>挑激动剂（化合物，这将导致活性的收缩），以使组织响应。 注：在大鼠胸主动脉，动脉平滑肌将积极签约，α-肾上腺素能受体激动剂由于该神经支配组织由交感神经系统。因为肾上腺素受体激动剂引起松弛肾上腺素能激动剂不会在肠组织非常有用。在这种情况下，也可以使用一个受体激动剂如乙酰胆碱（胆碱受体）。一种替代既福林和乙酰胆碱是使用非受体激动剂如高钾（K），为第一个挑战。在平滑肌，高钾操作来间接开放的钙通道，因而被视为平滑肌完整性的综合指数。 <ol><li>加入10 -5 M去氧向组织浴执行第一询问或唤醒挑战。为了达到此浓度的50毫升组织浴中，加50微升的10 -2 M溶液去氧肾上腺素。 注：50微升到50ml的PSS是一个1 / 1,000稀释，使最终的浓度比库存1000倍以下，或10 -5 M.内的组织浴中的知识量和一贯维护的体积的hamber是确定最终药物浓度是至关重要的。 </li></ol></li><li>添加10 -5 M去氧向组织浴，并允许收缩到峰值，然后高原，当数据的斜率变为零。确保记录每个实验活动，以帮助后的实验分析。 </li><li>高原后，排空和重新填充组织浴室新PSS彻底清洗激动剂出来。请务必记录这些事件也是如此。 注：经验法则是，激动剂在浴的浓度降低10倍每次洗涤。虽然，超过3-4次洗涤可能是必要的，以使所述组织回落到其平衡基线音（张力）。 </li><li>让组织其余基线的基调，然后再继续〜10分钟。 </li></ol> 7.实验注：哌唑嗪，α-肾上腺素受体拮抗剂，将被引入到组织浴室转移的浓度RESPONSE曲线去氧肾上腺素（PE）;这是可证明的拮抗作用。 <ol><li>到1浴中，添加车辆（H 2 O），其中哌唑溶解。到另一个，添加哌唑嗪的适当的浓度。在这种情况下，加入25微升车辆以一浴和25微升哌唑嗪的10 -5 M浓度的其他实现5×10 -9 M或在浴中的5nM的浓度。 注意：虽然除了车辆的在本实施例看来是不必要的，它使用具有潜在的是血管活性（ 例如，二甲基亚砜，乙醇，乙酸乙酯）其他车辆时必须这样做。添加相应的车辆即使没有证据存在它是血管活性。 </li><li>离开车辆/哌唑嗪在浴室和不洗组织1小时。 <ol><li>而组织的平衡，准备多个浓度激动剂（PE）的。包括一个宽范围的浓度，从浓度引发无反应（ 例如，&lt;/ em&gt;的10 -9 M 的 PE）到超过最大应答（浓度如 10 -4 M的PE）。自浓度-响应曲线是对数性的，从10 -1 1M贮存液使通过连续稀释六个单独的储备溶液的PE（10 -6 -10 -1 M）。确保所需要的各浓度的体积比三重所需的所有浴（ 即 &gt; 300微升）的总体积稍大。 </li></ol></li><li>着手加入激动剂，如表1所述。 注意：这个实验会产生累积浓度响应曲线PE;每次迭代考虑到物质的已加入到浴中的量。增加发生，直到他们不再增加收缩，或整个曲线趋于平稳。 <ol><li>添加每个药物浓度分别直到组织达到阈值。 </li><li>如果没有发生变化，添加在接下来的浓度系列; 见表2。 注意：这些添加的时机取决于所用的激动剂。例如，去甲肾上腺素和肾上腺素收缩的快速发展，并应在几分钟之内的高原。相比之下，内皮素-1收缩发展缓慢，不得高原近45分钟。其他的实验可以在组织进行施工的曲线，如本后，只要（1）该物质冲刷;和（2）它可以证明所述组织返回到正常收缩行为和不受先前刺激。 </li></ol></li></ol> 8.数据分析<ol><li>确保数据保存立即之前和之后的干预（ 例如，唤醒或全曲线）。 </li><li>找到并设置组织基线对每个组织浴室/输入通道，这将在数据分析提供帮助。 </li><li>一旦所述基线设置，发现对于每个浓度的最大反应，并记录瓒GE从基线。依次移动通过每个浓度并记录基线偏移。 </li><li>格拉夫将所得的数据。在任何图形程序做到这一点。 注意：图表垫棱镜设计用于药理学家，这是理想的这里提出的实验的类型。 </li></ol>

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M., O'Connor, C., McLoughlin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+changes+in+pH+on+wall+tension+in+isolated+rat+pulmonary+artery:+role+of+the+RhoA/Rho-kinase+pathway.">Effect of changes in pH on wall tension in isolated rat pulmonary artery: role of the RhoA/Rho-kinase pathway.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 287, L673-L684 (2004).</li><li>Tykocki, N. R., Thompson, J. M., Jackson, W. F., Watts, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ryanodine+receptors+are+uncoupled+from+contraction+in+rat+vena+cava.">Ryanodine receptors are uncoupled from contraction in rat vena cava.</a> Cell Calcium. 53, 112-119 (2013).</li></ol>

Authors have nothing to disclose.

的等长收缩力作为一种研究工具测量超过150年的历史，但它仍然是收缩组织2原型技术受体表征。该技术的功率是在其简单性和通用性：通过记录通过增加拮抗剂的存在或不存在激动剂的浓度引起的反应，信息无数可导出关于每种药物的药理学特性和受体向其中它结合3-5。这种类型的实验也争取有关的竞争与所使用的拮抗剂的非竞争性的信息，以及受体的异质性和非特异性药物作用6-8。因此，简单的排列组合，以这种孤立的组织浴实验，可以生成介导激动剂诱发肌肉收缩的受体的一个比较完整的药理学特性。 在激动方面，第在给定组织的激动剂电子相对效力（E MAX）和效力（EC 50）可被计算并与其它激动剂在相同组织1的响应。在我们的实验中，将大鼠胸主动脉孵育用载体或α1肾上腺素能受体拮抗剂哌唑嗪（5 nm）的一小时中添加α1肾上腺素能激动剂苯向组织浴并产生收缩之前。 图2给出修改的结果图1的，绿色的响应已被标以最大收缩由聚乙烯取得了明显的最大值，该半最大收缩标记为这样，然后与PE浓度相关联的原因造成的响应。确定这些值是识别实现了半最大（50％）的反应或EC 50值的激动剂的有效浓度。 不幸的是，使用激动剂依赖性参数独自一人来确定受体结合特性可并发9。理想的是，使用受体拮抗剂附加实验允许计算的两个重要参数是关键限定药物与受体之间的相互作用：所述拮抗剂离解常数（pK值B）的-log 10和摩尔拮抗剂的-log 10必要浓度以引发在浓度-反应曲线2倍向右移位（PA 2）10。无论K B＆和PA 2增益的一个事实，即他们是兴奋剂无关的值，保持不变，甚至不同组织11之间有用。从图2中的数据，PK B能够使用以下等式来计算，并根据欧共体别处计算50值： EC 50在没有哌唑嗪= 2×10 -8 M的 EC 50在预哌唑嗪SENCE = 7×10 -6 M 在下面的等式求解在K B： 登录（DR-1）=日志[B] -日志K B＆ 其中： [B] =拮抗剂浓度，或5×10 -9 M.登录（5×10 -9 M）= -8.3 DR =的EC 50值拮抗剂/ EC 50的情况下存在剂量率。如果有一个向右移位，则此值将大于1。因此： DR = 7×10 -6 M / 2×10 -8 M或700×10 -8 M / 2×10 -8 M =二分之七百= 350 代替[B]和DR： 登录（350-1）= -8 -登录K B＆ 2.54 = -8 -登录KB 的pK B = 10.54 该值哌唑嗪与获得的值一致时，哌唑嗪与α1肾上腺素RECEPT交互或12,13，这表明肾上腺素受体 ​​介导的收缩，以苯为α1肾上腺素能受体。 几个步骤是这些实验的成功是至关重要的。解剖后的组织必须保持在PSS，以防止组织的生存力的丧失。任何肌肉等长制剂，产生最大的力量对被动紧张14,15的最佳长度张力关系。在这个协议中所描述的实验中，最佳的被动张力是得到0.5克被动张力到组织的简要添加增量预先确定的。组织应该开始无音，然后平衡30分钟后，将组织用KCl（80毫米）的最大浓度，其产生的活动音调的挑战。然后，将组织洗涤并允许返回到基线音。 15分钟后，在基线，另一个0.5克被动张力置于与该过程重复，直到一个高原活性张力用另外的被动张力实现。在初步实验中，将4g总被动张力的测定，以达到最大活性的张力产生在主动脉环，因而张力这个总量前平衡放置在环。在程序上，这是最好的零之前的被动张力的应用，但可以在之前的实验中的任何时间进行。过度伸展的组织，在实验或夹层的任一点，将带负组织的生存力和实验的结果产生影响。放置组织在洗澡时，因为此步骤具有过度伸展的最高似然，这是最必要的。充分洗涤，在数量和持续时间，需要为可重现的效果。第二个挑战太快经过最初的挑战，日前组织已经回到基线紧张，会造成异常反应。如果就读可逆拮抗剂/抑制剂，组织不应该事先洗涤以激动剂另外，作为INHIBI器的浓度将降低。 有显着的优点和缺点，以分离的组织浴测定。 缺点：组织可能在钩上手术切除环或安置体验不同程度的损伤。由于内皮细胞是主动脉环的内衬，必须小心在放置在环上的挂钩，以便不破坏该细胞层。组织也可能有时间明显不同的长度，其中它们是可行的，在组织浴，而这必须被确定;不是所有的组织是相同的。沿着这些线路，组织可在全天的反应改变这样的时间控制成为了必要的控制每一个实验。这方面的一个很好的例子就是豚鼠气管，在一个典型的8小时的实验提高了收缩最大100％。药物是难溶于水的可沉淀出来的PSS。最后，一​​个累计第二药物是一种激动剂，可能会导致不同的结果，如果受体脱敏的激动剂发生的非累积加法;血管紧张素II是一种这样的药物，其示出快速快速耐受。 优点：其中的组织浴实验的主要优点是，它是实时;人们可以看到实验，因为它展现，并且可以迅速地作出结论，并在实验过程中规划后续步骤，以及故障排除。实验需要每天做。多个组织通常可以从一个动物制备，使得动物可以作为其自身的对照，而增加了强度以一个实验。人们也可以隔离所述组织从其他因素，以便测试该组织对药物的相对较纯的响应， 在体外实验中，如组织浴系统还允许使用相比， 在体内实验中少量的药物。 本文所描述的基本实验设计可以广泛修改，以允许更多的参数记录或引入其他外界刺激。例如，另外的电极允许支配神经16,17的电场刺激。通过加入热或pH探针，温度和pH值对收缩反应的影响也可以测量18,19。同样地，氧可以部分或全部用N 2以评估缺氧诱导的作用取代。此外，可以使用等距收缩的测量在此视频中使用的相同的基本原理来开发系统，允许等长张力的发展和变化的伴测量细胞内钙20。信号转导也容易了研究，由于系统存在的响应时能迅速冷冻组织样本，使得一个信号转导途径体系的活性可以生物化学进行验证。 EQU的变化ipment可以用来做，这是巨大的。这整个系统，建造或自动左右手，可以从多个不同的公司购买。在这个协议中使用的组织浴和组织持有人手工吹制（组织浴）和手工建造（持有人）通过一个内部的密歇根州立大学机械车间。

药理学学科采用了分离组织浴系统超过150年。该系统的多功能性使得科学家在世界各地以表征受体和受体信号转导，这方面的知识形成已处理的数以百万计的疾病或病症，例如高血压，心脏衰竭，糖尿病，胃肠道疾病，膀胱的个体的疗法的基础功能障碍，哮喘和吞咽障碍，仅举几例。为了这一天，分离的组织浴保持药物开发和基础研究的一个重要方面，因为它允许所述组织以用作组织。在此朱庇特的课，正式的协议共享演示视觉和虚拟实验，从一个孤立的组织浴实验，测量等长收缩，这使得受体的表征利用数据。 该技术的主要优点在于，所述组织是过着第二功能为一体的组织，用生理结果（收缩或松弛）是相关于身体。它是一种合成的步骤（药物 - 受体相互作用，信号传导，第二信使的产生，改变平滑肌兴奋性，并改变组织功能）。而其它技术允许每个步骤的研究（ 例如，放射性配体结合药物的亲和力，第二信使测定）中，分离的组织浴技术允许集成的所有这些步骤1。另一个优点是，保持组织功能允许的重要的药理学变量是在一个组织中的VS的蜂窝环境更有意义的计算;它更接近如何检查药品将在工体作为一个整体。

<table border="0" cellpadding="0" cellspacing="0" width="863">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Name of Material/ Equipment</td>
			<td style="width:203px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LabChart Software</td>
			<td>ADInstruments</td>
			<td></td>
			<td>7.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PowerLab (4 channel)&#160;</td>
			<td>ADInstruments</td>
			<td>ML760</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuadBridge (4 channel)</td>
			<td>ADInstruments</td>
			<td>ML112</td>
			<td>ML112</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Grass Adapter Cable</td>
			<td>ADInstruments</td>
			<td>MLAC11</td>
			<td>MLAC11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Grass Force-Displacement Transducer</td>
			<td>Grass Instrument Co</td>
			<td>FT03</td>
			<td>FT03</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Grass Transducer Cable</td>
			<td>Grass Instrument Co</td>
			<td>TAC-7 REV-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BNC to BNC Cable</td>
			<td>ADInstruments</td>
			<td>MLAC01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IsoTemp 2100</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>IC-2100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Bath</td>
			<td>Multiple Sources&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Physiological Salt Solution</td>
			<td></td>
			<td></td>
			<td>PSS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Braided Silk Suture</td>
			<td>Harvard Apparatus</td>
			<td>51-7615</td>
			<td>SP104</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ring Stand</td>
			<td>Humboldt MFG Co</td>
			<td>H-2122 7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting Dishes</td>
			<td>Handmade with Silicone</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tygon Tubing</td>
			<td>VWR Scientific</td>
			<td>63010-100</td>
			<td>R-3603</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hose Clamps</td>
			<td>Cole-Parmer Instrument Co</td>
			<td>06832-08</td>
			<td>SNP-8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50ml Muscle Bath</td>
			<td>Eberhartglass Blowing</td>
			<td>Custom</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250ml Warming Chambers</td>
			<td>Eberhartglass Blowing</td>
			<td>Custom</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gas Dispersion Tube</td>
			<td>Ace Glass</td>
			<td>7202-06</td>
			<td>7202-02</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micrometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Custom Stands</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Three-Prong Clamps</td>
			<td>VWR International</td>
			<td></td>
			<td>Talon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">S-Connector</td>
			<td>VWR International</td>
			<td></td>
			<td>Talon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Hooks</td>
			<td>Hand Made in House</td>
			<td>Custom</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Dissection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica Stereomicroscope MZ6</td>
			<td>Leica</td>
			<td></td>
			<td>10447254</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereomaster Microscope Fiber-Optic Light Source</td>
			<td>Fisher Scientific</td>
			<td>12562-36</td>
			<td>12562-36</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Culture Petri Dish</td>
			<td>Pyrex</td>
			<td></td>
			<td>7740 Glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sylgard Silicone Elastomer</td>
			<td>Dow Corning</td>
			<td></td>
			<td>Sylgard 170 Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vannas Scissors</td>
			<td>George Tiemann &#38; Co</td>
			<td>160-150</td>
			<td>160-150</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Splinter &#38; Fixation Forceps</td>
			<td>George Tiemann &#38; Co</td>
			<td>160-55</td>
			<td>160-55</td>
		</tr>
	</tbody>
</table>

measurement of smooth muscle function in the isolated tissue bath-applications to pharmacology research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

在激动而言，在给定组织的激动剂的相对效力（E MAX）和效力（EC 50）可被计算并与其它激动剂在相同组织1的响应。在我们的实验中，将大鼠胸主动脉孵育用载体或α1肾上腺素能受体拮抗剂哌唑嗪（5 nm）的一小时中添加α1肾上腺素能激动剂苯向组织浴，并产生收缩（ 图1）之前。 图2呈现改性图1的结果，绿色响应有显着的最大收缩由聚乙烯取得了明显的最大值，该半最大收缩标记为这样，然后与PE浓度相关联的原因造成的响应。确定这些值是识别实现了半最大（50％）的反应或EC激动剂的有效浓度<sUB&gt; 50值。这是donegraphically在使用可用于曲线EC 50值的拟合和计算逻辑函数为S形曲线的这个example.Computer软件。 在产生和解释这些结果，使用激动剂的低和高浓度来创建浓度 - 响应曲线是重要的。如果没有足够的积分以示稳定的基线和最高的高原，EC 50和E MAX只能估计。这是在本实施例以图形方式进行。使用逻辑函数为S形曲线的计算机软件，可用于曲线EC 50值的拟合和计算。 <img alt="图1" src="/files/ftp_upload/52324/52324fig1highres.jpg" /> 图1：组织浴示意图卡通代表组织的环放置在双壁，水夹套组织巴日。缓冲流入和流出由一个3路玻璃阀集成到室的底部调节。 的 O 2 / CO 2通过该密封用垫片，以防止PSS或气体的泄漏的曝气装置中鼓泡。再循环输入低于以保持适当的再循环和防止形成气穴会导致温度失调的输出。 <img alt="图2" src="/files/ftp_upload/52324/52324fig2highres.jpg" /> 图2：大鼠胸主动脉内皮+上图描绘了四个独立的实验（不同的动物并通过在由不同的颜色代表不同的一组窗口不同研究者进行），以测试哌唑嗪的转移在PE诱导的收缩的能力。哌唑嗪（5纳米）清楚地错开的PE诱导的收缩曲线向右以并行方式。 图3：大鼠胸主动脉内皮+ EC 50值的PE图形估计在不存在（载体）和拮抗剂的存在下（哌唑嗪）。绿线（C组）只被标记。 <table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td> 盐 </td><td> MW（克/摩尔） </td><td> 5升 </td><td> FINAL毫米 </td></tr><tr><td></td><td></td><td></td><td> （克） </td></tr><tr><td>氯化钠</td><td> 58.45 </td><td> 37.99 </td><td> 130 </td></tr><tr><td>氯化钾</td><td> 74.56 </td><td> 1.75 </td><td> 4.7 </td></tr><tr><td> KH2PO4 </td><td> 136.1 </td><td> 0.8 </td><td> 1.18 </td></tr><tr><td>硫酸镁•7H 2 O </td><td> 246.5 </td><td> 1.45 </td><td> 1.17 </td></tr><tr><td>碳酸氢钠</td><td> 84.21 </td><td> 6.25 </td><td> 14.9 </td></tr><tr><td>葡萄糖</td><td> 180.16 </td><td>五</td><td> 5.5 </td></tr><tr><td> EDTA </td><td> 380 </td><td> 0.05 </td><td> 0.03 </td></tr></tbody></table> 表1：正常生理盐溶液（PSS）的配方在本实验中所用的碳酸氢盐缓冲的PSS的含量。包括的是所有的盐的分子量，并加入到5升的dh 2 O，以获得所需浓度的量。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 在浴浓度 </td><td> 除了 ​​激动剂洗澡做 </td></tr><tr><td> 1×10 -9 M的 </td><td> 50毫升1×10 -6 M的 </td></tr><tr><td> 3×10 -9 M的 </td><td>百毫升1×10-6 M </td></tr><tr><td> 1×10 -8 M的 </td><td> 35毫升1×10 -5 M </td></tr><tr><td> 3×10 -8 M的 </td><td>百毫升1×10 -5 </td></tr><tr><td> 1×10 -7 M的 </td><td> 35毫升1×10 -4 M </td></tr><tr><td> 3×10 -7 M的 </td><td>百毫升1×10 -4 M </td></tr><tr><td> 1×10 -6 M的 </td><td> 35毫升1×10 -3 M </td></tr><tr><td> 3×10 -6 M的 </td><td>百毫升1×10 -3 M </td></tr><tr><td> 1×10 -5 M </td><td> 35毫升1×10 -2 M </td></tr><tr><td> 3×10 -5 M </td><td>百毫升1×10 -2 M </td></tr><tr><td> 1×10 -4 M </td><td> 35毫升1×10 -1 M </td></tr></tbody></table> 表2：组织浴添加剂激动剂浓度要加入激动剂的量，以每一个浴中以亲perly构造累积浓度 - 响应曲线。所需的浴浓度是通过加入增加激动剂的浓度的连续量来实现的。每次加入考虑激动剂已经存在于浴中的浓度。这样做是为了最大限度地减少在组织浴中，会导致使用激动剂的单一浓度的体积增加的增加量。

Watch this Scientific Journal Video about Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research at JoVE.com

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

平滑肌功能中分离出来的组织浴，应用药理学研究测量

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

measurement-smooth-muscle-function-isolated-tissue-bath-applications

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Biology

Unit 1

Drugs Acting on Autonomic Nervous System: Adrenergic Agonists and Antagonists Agents

SCIENTISTS IN ACTION

135.8K 次观看.  Michigan State University. This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition. 

视频: Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

焦距钙 2 +</sup瞬态检测平滑肌

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

科研

生物学

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.
The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. 
The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However 
curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, 
detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable 
preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting 
cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. 
The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. 
It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet 
for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle 
from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it 
continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a 
genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of 
calcium signaling in the arteriolar endothelium.

A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.

鼠标提睾肌肌肉制备的微循环活体成像

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营：从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

体外评估对离体骨骼肌的收缩，疲劳性和交替“

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

探讨动脉平滑肌的KV7钾离子通道功能使用膜片钳电生理和压力Myography

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

在收缩力测量评估肌肉功能在斑马鱼幼虫

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

细胞内钙的双光子成像<sup&gt; 2+</sup&gt;的离体大鼠主动脉的处理和一氧化氮的内皮细胞和平滑肌细胞

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

基于FRET钙内波动培养平滑肌细胞使用的肌质网的测量共焦成像

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

鼠冠状动脉血管平滑肌细胞的分离

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

鼠标离体肾技术

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...