Name: measurement of antibody effects on cellular function of isolated cardiomyocytes
Uploaded: 2013-03-08T12:00:00
Description: Watch this Scientific Journal Video about Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes at JoVE.com

这项工作是支持的Sonderforschungsbereich Transregio 19（SFB/TR19，C2），德意志研究联合会（DFG），联邦经济和技术项目ZIM-KF 2727801MD0和创新能力的中心 - 体液免疫反应在心血管疾病（ ZIK加息FKZ 03Z2CN12，BMBF）的联邦教育和研究部，德国。房屋和实验动物学会实验动物科学（促进协会毛皮Versuchstierkunde，GV-SOLAS）和联邦的欧洲实验动物科学协会（FELASA“）的建议进行。

1。抗体从患者标本分离<ol><li>准备迷你免疫吸附柱填充3毫升2毫升病人EDTA血浆抗IgG琼脂糖每到一个空的Econo豆列。 IgG抗体隔离需要至少2毫升的病人血浆中。 </li><li>将抗-IgG琼脂糖凝胶的顶部上的过滤器，剖开列的前端下部，并按下的过滤器可达到约1滴/秒的流速。让保存液用完的列。 </li><li>洗柱用3倍体积的0.9％NaCl中，每体积的血浆。 </li><li>移液器的血浆列。当等离子体已完全沉浸在抗-IgG琼脂...

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所提出的方法提供了一种合适的方法来检测自身抗体在DCM来源不明的患者的心脏功能有效。在与其他方法相比， 例如：对β1-肾上腺素能受体的功能活性的抗体的检测由他们影响cAMP水平6，我们的方法是独立于一个特定的表位。当然也有其他的文献中描述的表位独立分析， 即计算跳动的乳鼠心肌细胞13，在成人的心肌细胞钙电流测量膜片钳技术或孤立的浦肯野纤维的?...

<table border="1"><tbody><tr><td> 的试剂/设备名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> Immunosorba保护解决方案</td><td>费森尤斯医疗保健</td><td> 903609211 </td><td></td></tr><tr><td>型胶原酶2 </td><td>电池系统</td><td> LS004176 </td><td></td></tr><tr><td> BSA，无脂肪酸</td><td> Sigma-Aldrich公司</td><td> A6003 </td><td></td></tr><tr><td>层粘连蛋白</td><td> Sigma-Aldrich公司</td><td> L2020 </td><td></td></tr><tr><td>的Fura-2 AM </td><td> Sigma-Aldrich公司</td><td> F0888 </td><td>在DMSO中的1毫克/毫升</td></tr><tr><td>的Econo PAC色谱柱</td><td> BioRad公司</td><td> 732-1010 </td><td></td></tr><tr><td> SPECTRA / POR生物纤维素酯透析膜上Ë </td><td> Spectrumlabs </td><td> 131417 </td><td></td></tr><tr><td> 4井腔室盖玻片</td><td> Nunc公司</td><td> 155383 </td><td></td></tr><tr><td>心肌细胞的钙和收缩录音系统</td><td> IonOptix </td><td></td><td></td></tr><tr><td> IonWizard 6.0分析软件</td><td> IonOptix </td><td></td><td></td></tr></tbody></table>

measurement of antibody effects on cellular function of isolated cardiomyocytes

扩张型心肌病（DCM）是在年轻的成年人1心脏衰竭的主要原因之一。虽然遗传倾向和博览会的有毒物质被称为的起源DCM，约三分之一的患者对本病的原因，在很大程度上仍然不清楚。在这些患者中有相当多的，已经对心脏表位抗体检测和怀疑本病的发生和发展2,3中发挥了举足轻重的作用。心脏自身抗体的重要性，强调消除自身抗体的免疫吸附3-5后，在DCM患者血流动力学的改善。已经被确定各种特异性抗原2,3和针对这些目标的抗体，可以通过免疫测定法检测。然而，这些方法不能区分刺激（及因此功能有效）和阻断自身抗体。有越来越多的evidenCE，这种区别是非常重要的6,7。它也可以被假定，若干心性抗体的目标仍然是身份不明，因此不能检测由免疫测定法。因此，我们建立了功能活性的心性的抗体，独立的各自的抗原的检测方法。用于该方法的背景是通常观察到的功能区域心脏蛋白在哺乳动物8,9之间的高同源性。这表明，心脏针对人的抗原的抗体与非人类的靶细胞，它允许测试从DCM患者对成年大鼠心肌细胞的IgG交叉反应。我们的方法包括3个步骤：第一，IgG抗体是从病人的血浆，使用琼脂糖凝胶偶联的抗-IgG抗体，从免疫吸附的列（德国，Teterow PlasmaSelect）得到分离。二，成人的心肌细胞分离胶原酶灌注在Langendorff灌流装置，使用AP议定修改以前的作品10,11。将得到的心肌细胞的附着层粘连蛋白涂层的腔coverglasses和染色的Fura-2，钙的选择性荧光染料，该染料可以容易地进入细胞，观察细胞内钙（Ca 2 +）含量12。在最后一步中，病人的IgG的效果上的细胞缩短和Ca 2 +瞬变字段刺激心肌在线监视使用市售的细胞钙和收缩监测系统（IonOptix，弥尔顿，MA，USA）连接到一个标准的逆荧光显微镜。

两个例子为测量在该领域的蜂窝性收缩力刺激成人的心肌细胞（ 图2），使用下面给出的心肌细胞的Ca 2 +和收缩记录系统， 图3给出了一个控制测量的印象，而在图4中的效果cardiodepressive抗体的病人，在DCM中所示。 在这两个例子中，初始细胞缩短和Ca 2 +瞬态期间灌流与EB表现出非常明确和一贯的山峰，这是一个先决条件的?...

Watch this Scientific Journal Video about Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes at JoVE.com

Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

所提出的方法提供了一种方法扩张型心肌病的患者血浆中的检测功能有效的心性自体抗体，不论特异性抗原，通过分析隔离病人免疫球蛋白对大鼠离体心肌细胞的缩短及细胞内钙瞬变的影响。

测量抗体乳鼠心肌细胞对细胞功能的影响

Dilated cardiomyopathy  (DCM) is one of the main causes for heart failure in younger adults1.  Although genetic disposition and exposition to toxic ...

Research

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Immunology and Infection

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

胞内钙的测量 2 +</sup在隔离的收缩淋巴管

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

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