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琼脂糖凝胶，用相同体积0.9％NaCl溶液洗。 </li><li>吸取1，pH值为2.8的0.2M甘氨酸盐酸血浆体积上的列，并让沉浸到琼脂糖。添加另一个卷甘氨酸盐酸列上。收集的流量，通过在一个15毫升的试管中，将pH调节至7.3，用0.5M的Tris-HCl，pH值8.0。 </li><li>若要重新列，用3个血浆容量甘氨酸盐酸。用2倍体积的0.9％NaCl的最终洗涤后，这个字段可以被用于下一个血浆样品。可替代地，列可以填充与保存溶液，并存储在4 - 8℃下进行最多达4周。 </li><li>对于心肌细胞上使用，所收集的IgG级分透析实验缓冲液（EB）。对于制备的EB，添加0.6毫摩尔MgCl 2，117 mM氯化钠，2.8 mM KCl中，1.2毫摩尔KH 2 PO 4，1.2毫摩尔的CaCl 2，10mM HEPES和20mM葡萄糖的1升蒸馏水，在磁力搅拌器上，并调整pH值7.3。 </li><li>一种纤维素酯渗析膜管中填充的IgG级分，具有分子量100 kDa的切断。将管在1升的EB，并用磁力搅拌器缓慢搅拌，在4℃下8小时后，在透析管转移到3升的新鲜的EB和另一透析20小时，在4℃。 </li><li>经过透析，加热样品30分钟，在57℃下在水浴中以inactivatË补体因子。确定总IgG浓度，并准备含330微克的IgG各等份。当心肌细胞样品的测量（4.3），填补了EB的1毫升等分试样。可以存储未稀释的等分试样在-20℃直至进一步使用。 </li></ol> 2。大鼠心肌细胞的分离<ol><li>对于制备的Ca 2 +的隔离缓冲器（IB），添加110 mM氯化钠，2.6 mM KCl中，1.2 mM的用MgSO 4，1.2毫摩尔KH 2 PO 4，20mM的HEPES，11mM葡萄糖1升蒸馏水中，使用磁力搅拌器。调节pH至7.4，在室温和过滤器通过0.2μm瓶顶过滤器进行灭菌。 </li><li>填充80毫升Langendorff灌流恒温系统上水库IB。调整恒温器设置，以保持在37℃的温度的缓冲和曝气用95％O 2/5％CO 2的 30分钟的缓冲区。 </li><li>称取约10,000 - 12,000 U的collagenase II型，175毫克无脂肪酸BSA和溶解在20毫升IB含有22.5微升的100mM CaCl 2的原液。控制隔离缓冲液的pH值在上水库和如果需要的话进行调整。 </li><li>只Wistar大鼠175 - 200克，375微克/公斤体重硫喷妥钠麻醉。 2500 IU肝素以硫喷妥钠解决方案。开胸手术后，切除心小心地用一个完整的主动脉弓，并将其转移到冰冷的0.9％的氯化钠。清洁对心脏的血液和周围组织和转移到新鲜的0.9％NaCl溶液中。暴露主动脉，打开节流的Langendorff系统，获得滴的速度2-3/sec的心脏的套管。 </li><li>最多3分钟，流速为1滴/秒，洗出剩余的血灌注心脏。开始流通的Langendorff系统中的IB。从上游水库中取出20毫升缓冲液，填补在的胶原酶溶液和填充的缓冲，需要达到80毫升。循环的c<html>另一个27分钟ollagenase溶液。流速应周期性地控制和维持在1滴/秒使用节流。 </li><li>从套管分离的心，干净的心房和主动脉，切成小块。允许在的胶原酶溶液运行到下水库，降低音量到最大40毫升和进一步消化切碎的心室为10 - 15分钟，在连续曝气与95％O 2/5％CO 2。之后，过滤该细胞悬浮液通过200μm的筛目大小的纱布入50毫升的管。 </li><li>还原的Ca 2 +的含量的细胞在3个步骤：在43×g离心2分钟，在室温和重悬细胞在10ml IB含有200μMCaCl 2的离心细胞悬浮液。再次离心，重悬细胞与500μM 氯化钙 10毫升IB。经过最后的离心重悬心肌在无菌过滤EB（见1.8），50000个细胞/ ml的密度。 </li></ol>e_title“&gt; 3。Fura-2的染色中的心肌细胞<ol><li>大衣4腔室盖玻片，每孔10微克层粘连蛋白和等待，直到它完全干燥。 </li><li>导入各孔中，切割尖端用微量填充1毫升上述心肌细胞的悬浮液。允许在室温下的1小时的细胞附着。 </li><li>稀释10微升的1毫克/毫升的Fura-2 AM在5毫升的EB的原液。保持在黑暗中的染色溶液。 </li><li>脱掉缓冲液和未连接的细胞从盖玻片和吸移管0.5毫升到各孔中的染色溶液。 10分钟适度的摇晃下培养细胞。随后，起飞染色液，洗细胞一次，用1毫升EB终于填补0.5毫升的EB中，每孔。在染色过程中应避免直射阳光，并始终保持在黑暗中染色细胞。 </li></ol> 4。细胞缩短和Ca 2 +瞬态记录<ol><li>将腔室盖玻片的倒数荧光米icroscope连接到一个心肌细胞的钙和收缩记录系统。一个定制的电极与第一井的流入和流出管的位置。 superfuse心肌细胞与1毫升/分钟的EB和开始电刺激（1赫兹，20伏，5毫秒的持续时间）。使细胞适应约2分钟的刺激。 </li><li>选择一个完整的棒状清晰的条纹和不断收缩的心肌细胞。的视场中的单元格的中心位置，并打开相机通道。取向的细胞内水平的视频区，通过旋转的小区成帧适配器和移动的显微镜载物台上。 </li><li>调整视频区域的边缘检测控制单元（ 图2），打开荧光通道和创纪录的10 - 15宫缩获得初始细胞缩短和Ca 2 +瞬态。 </li><li>关闭显微镜的光通道，以避免漂白的荧光染色。切换流过的样品从EB绕过了3路阀和superfuse心肌细胞用1ml的患者的IgG。停泵前空气达到良好。 </li><li>打开光通道，并记录10 - 15收缩获得的急性细胞反应。暂停录制，光通道再次关闭。 2和5分钟后重复记录。 </li><li>取出电极的阱。彻底清洁管，并继续与EB的下一个良好的腔室盖玻片。 </li><li>细胞缩短和Ca 2 +瞬变与IonWizard软件使用“Edge-Length/Length”和“呋喃-2 -数字/比例减去”窗口，分别分析。计算从初始细胞缩短和Ca 2 +瞬时用于急性称为基线（提单％峰值小时）的峰高，2分钟和5分钟响应的百分比变化。 </li></ol>

<ol>
	<li>Roger, V. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+disease+and+stroke+statistics--2011+update:+a+report+from+the+American+Heart+Association.">Heart disease and stroke statistics--2011 update: a report from the American Heart Association.</a> Circulation. 123 (4), e18 (2011).</li><li>Caforio, A. L., Vinci, A., Iliceto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-heart+autoantibodies+in+familial+dilated+cardiomyopathy.">Anti-heart autoantibodies in familial dilated cardiomyopathy.</a> Autoimmunity. 41 (6), 462 (2008).</li><li>Yoshikawa, T., Baba, A., Nagatomo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+mechanisms+underlying+dilated+cardiomyopathy.">Autoimmune mechanisms underlying dilated cardiomyopathy.</a> Circ. J. 73 (4), 602 (2009).</li><li>Felix, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+effects+of+immunoadsorption+and+subsequent+immunoglobulin+substitution+in+dilated+cardiomyopathy:+Three-month+results+from+a+randomized+study.">Hemodynamic effects of immunoadsorption and subsequent immunoglobulin substitution in dilated cardiomyopathy: Three-month results from a randomized study.</a> Journal of the American College of Cardiology. 35 (6), 1590 (2000).</li><li>Staudt, A., et al. <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+immunoglobulin+G3+subclass+in+dilated+cardiomyopathy:+results+from+protein+A+immunoadsorption.">Role of immunoglobulin G3 subclass in dilated cardiomyopathy: results from protein A immunoadsorption.</a> Am. Heart J. 150 (4), 729 (2005).</li><li>Nikolaev, V. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+fluorescence+method+for+the+rapid+detection+of+functional+beta1-adrenergic+receptor+autoantibodies+in+heart+failure.">A novel fluorescence method for the rapid detection of functional beta1-adrenergic receptor autoantibodies in heart failure.</a> J. Am. Coll. Cardiol. 50 (5), 423 (2007).</li><li>Jahns, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibodies+activating+human+beta1-adrenergic+receptors+are+associated+with+reduced+cardiac+function+in+chronic+heart+failure.">Autoantibodies activating human beta1-adrenergic receptors are associated with reduced cardiac function in chronic heart failure.</a> Circulation. 99 (5), 649 (1999).</li><li>Gocayne, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+structure+of+rat+cardiac+beta-adrenergic+and+muscarinic+cholinergic+receptors+obtained+by+automated+DNA+sequence+analysis:+further+evidence+for+a+multigene+family.">Primary structure of rat cardiac beta-adrenergic and muscarinic cholinergic receptors obtained by automated DNA sequence analysis: further evidence for a multigene family.</a> Proceedings of the National Academy of Sciences. 84 (23), 8296 (1987).</li><li>Jaenicke, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complete+sequence+of+the+human+beta-myosin+heavy+chain+gene+and+a+comparative+analysis+of+its+product.">The complete sequence of the human beta-myosin heavy chain gene and a comparative analysis of its product.</a> Genomics. 8 (2), 194 (1990).</li><li>Piper, H. M., Volz, A., Schwartz, P., Piper, 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=.">.</a> Cell Culture Techniques in Heart and Vessel Research. , (1990).</li><li>Kubin, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+endothelial+cells+remodel+cultured+adult+cardiomyocytes+and+increase+their+survival.">Microvascular endothelial cells remodel cultured adult cardiomyocytes and increase their survival.</a> Am. J. Physiol. 276 (6 Pt. 2), H2179 (1999).</li><li>Grynkiewicz, G., Poenie, M., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+generation+of+Ca2++indicators+with+greatly+improved+fluorescence+properties.">A new generation of Ca2+ indicators with greatly improved fluorescence properties.</a> J. Biol. Chem. 260 (6), 3440 (1985).</li><li>Magnusson, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmunity+in+idiopathic+dilated+cardiomyopathy.+Characterization+of+antibodies+against+the+beta+1-adrenoceptor+with+positive+chronotropic+effect.">Autoimmunity in idiopathic dilated cardiomyopathy. Characterization of antibodies against the beta 1-adrenoceptor with positive chronotropic effect.</a> Circulation. 89 (6), 2760 (1994).</li><li>Wakefield, I. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+application+of+in+vitro+methods+to+safety+pharmacology.">The application of in vitro methods to safety pharmacology.</a> Fundam. Clin. Pharmacol. 16 (3), 209 (2002).</li><li>Kajzar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+physiological+conditions+for+cell+analyses:+forces+of+heart+muscle+cells+suspended+between+elastic+micropillars.">Toward physiological conditions for cell analyses: forces of heart muscle cells suspended between elastic micropillars.</a> Biophys. J. 94 (5), 1854 (2008).</li></ol>

没有利益冲突的声明。

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?...

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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 ...

measurement-antibody-effects-on-cellular-function-isolated

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Immunology and Infection

11.8K 次观看.  University Medicine Greifswald. 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。

视频: Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses1. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation2. This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency4. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively5. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.
Contraction of smooth muscle is generally regulated by the cytosolic Ca2+ concentration ([Ca2+]i) plus sensitivity to Ca2+, of the contractile elements in response to changes in the environment surrounding the cell6. [Ca2+]i is determined by the combination of the movement of Ca2+ through plasma membrane ligand or voltage gated Ca2+ channels and the release and uptake of Ca2+ from internal stores. Cytosolic Ca2+ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction8. However, the sensitivity of this pathway to Ca2+ can be regulated by the MLC phosphatase (MLCP)9. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.
Here, we present a method to evaluate changes in [Ca2+]i over time in isolated, perfused lymphatics in order to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca2+]i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca2+]i was determined by loading lymphatics with the ratiometric, Ca2+-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction.

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 ...

科研

免疫与感染

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

微孔板的检测，以评估RBL-2H3肥大细胞脱颗粒：三氯生的影响不使用有机溶剂的化学效应

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

定量检测抗体诱导补体激活的方法对红血细胞

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

对白细胞从流程的分析招聘基质细胞的影响

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

粒子结合Bet v 1的变应原PM10的流式细胞分析

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

肽扫描辅助一个单克隆抗体识别的线性B细胞表位的鉴定

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

腹腔葡萄糖耐受试验、肺功能测定和肺固定研究肥胖和代谢障碍对肺部结局的影响

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

抗体药物对小鼠骨髓及腹腔巨噬细胞活化作用的评价

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

小鼠涎腺功能的测定方法

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb development since its first report in 1975. As an alternative technology, phage display methods for mAb development are increasingly attractive since Humira, the first phage-derived antibody and one of the best-selling mAbs, was approved for clinical treatment of rheumatoid arthritis in 2002. As a non-animal based mAb development technology, phage display bypasses antigen immunogenicity, humanization, and animal maintenance that are required from traditional hybridoma technology based antibody development. In this protocol, we describe a method for construction of synthetic phage-displayed Fab libraries with diversities of 109-1010 obtainable with a single electroporation. This protocol consists of: 1) high-efficiency electro-competent cell preparation; 2) extraction of uracil-containing single-stranded DNA (dU-ssDNA); 3) Kunkel&#39;s method based oligonucleotide-directed mutagenesis; 4) electroporation and calculation of library size; 5) protein A/L-based enzyme-linked immunosorbent assay (ELISA) for folding and functional diversity evaluation; and 6) DNA sequence analysis of diversity.

This protocol describes a detailed procedure for the construction of a phage-displayed synthetic antibody library with tailored diversity. Synthetic antibodies have broad applications from basic research to disease diagnostics and therapeutics.

具有量身定做多样性的合成噬菌体展示库的构建

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb ...