Name: high-throughput flow cytometry cell-based assay to detect antibodies to n-methyl-d-aspartate receptor or dopamine-2 receptor in human serum
Uploaded: 2013-11-23T14:00:00
Description: Watch this Scientific Journal Video about High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum at JoVE.com

这项工作是由澳大利亚国家卫生和医学研究理事会，星科基金会（澳大利亚），抽动秽语综合征协会（美国）的支持，该崔西多发性硬化症研究基金会和多发性硬化症研究澳大利亚，Petre的基金会（澳大利亚），丽贝卡L。库珀医学研究基金会（澳大利亚）。我们感谢所​​有的病人，谁提供的样品为我们研究家族成员。

<html> 1。亚克隆策略构建的pIRES2-EGFP载体编码D2R或NMDA受体<ol><li>获得人类D2R或NMDA受体亚单位1（NR1）的全长cDNA克隆。 </li><li>选择的表达载体，如质粒pIRES2-EGFP，这是适合于跨膜蛋白，具有增强的绿色荧光蛋白在内部核糖体进入位点（IRES）的控制（GFP）的报告，使两种抗原和GFP中被共表达的表达细胞分开。 </li><li>内的pIRES2-EGFP载体亚克隆人cDNA。 <ol><li>亚克隆的cDNA，用适当的限制性内切酶（例如...

<ol>
	<li>Lancaster, E., Martinez-Hernandez, E., Dalmau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encephalitis+and+antibodies+to+synaptic+and+neuronal+cell+surface+proteins.">Encephalitis and antibodies to synaptic and neuronal cell surface proteins.</a> Neurol. 77, 179-189 (2011).</li><li>Vincent, A., Bien, C. G., Irani, S. R., Waters, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibodies+associated+with+diseases+of+the+CNS:+new+developments+and+future+challenges.">Autoantibodies associated with diseases of the CNS: new developments and future challenges.</a> Lancet Neurol. 10, 759-772 (2011).</li><li>Dalmau, 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=Anti-NMDA-receptor+encephalitis:+case+series+and+analysis+of+the+effects+of+antibodies.">Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies.</a> Lancet Neurol. 7, 1091-1098 (2008).</li><li>Dalmau, 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=Paraneoplastic+anti-N-methyl-D-aspartate+receptor+encephalitis+associated+with+ovarian+teratoma.">Paraneoplastic anti-N-methyl-D-aspartate receptor encephalitis associated with ovarian teratoma.</a> Ann Neurol. 61, 25-36 (2007).</li><li>Dale, R. C., 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=N-methyl-D-aspartate+receptor+antibodies+in+pediatric+dyskinetic+encephalitis+lethargica.">N-methyl-D-aspartate receptor antibodies in pediatric dyskinetic encephalitis lethargica.</a> Ann Neurol. 66, 704-709 (2009).</li><li>Lancaster, E., 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=Antibodies+to+the+GABA(B)+receptor+in+limbic+encephalitis+with+seizures:+Case+series+and+characterisation+of+the+antigen.">Antibodies to the GABA(B) receptor in limbic encephalitis with seizures: Case series and characterisation of the antigen.</a> Lancet Neurol. 9, 67-76 (2010).</li><li>Lai, M., 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=AMPA+receptor+antibodies+in+limbic+encephalitis+alter+synaptic+receptor+location.">AMPA receptor antibodies in limbic encephalitis alter synaptic receptor location.</a> Ann Neurol. 65, 424-434 (2009).</li><li>Irani, S. 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=Antibodies+to+Kv1+potassium+channel-complex+proteins+leucine-rich,+glioma+inactivated+1+protein+and+contactin-associated+protein-2+in+limbic+encephalitis,+Morvan&#8217;s+syndrome+and+acquired+neuromyotonia.">Antibodies to Kv1 potassium channel-complex proteins leucine-rich, glioma inactivated 1 protein and contactin-associated protein-2 in limbic encephalitis, Morvan&#8217;s syndrome and acquired neuromyotonia.</a> Brain. 133, 2734-2748 (2010).</li><li>Lai, M., 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=Investigation+of+LGI1+as+the+antigen+in+limbic+encephalitis+previously+attributed+to+potassium+channels:+a+case+series.">Investigation of LGI1 as the antigen in limbic encephalitis previously attributed to potassium channels: a case series.</a> Lancet Neurol. 9, 776-785 (2010).</li><li>Lancaster, E., 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=Antibodies+to+metabotropic+glutamate+receptor+5+in+the+Ophelia+syndrome.">Antibodies to metabotropic glutamate receptor 5 in the Ophelia syndrome.</a> Neurol. 77, 1698-1701 (2011).</li><li>Dale, R. C., 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=Antibodies+to+surface+dopamine-2+receptor+in+autoimmune+movement+and+psychiatric+disorders.">Antibodies to surface dopamine-2 receptor in autoimmune movement and psychiatric disorders.</a> Brain. , (2012).</li><li>Titulaer, M. 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=Treatment+and+prognostic+factors+for+long-term+outcome+in+patients+with+anti-NMDA+receptor+encephalitis:+an+observational+cohort+study.">Treatment and prognostic factors for long-term outcome in patients with anti-NMDA receptor encephalitis: an observational cohort study.</a> Lancet Neurol. 12, 157-165 (2013).</li><li>Hughes, E. G., 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=Cellular+and+synaptic+mechanisms+of+anti-NMDA+receptor+encephalitis.">Cellular and synaptic mechanisms of anti-NMDA receptor encephalitis.</a> J Neurosci. 30, 5866-5875 (2010).</li><li>Bien, C. G., 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=Immunopathology+of+autoantibody-associated+encephalitides:+clues+for+pathogenesis.">Immunopathology of autoantibody-associated encephalitides: clues for pathogenesis.</a> Brain. 135, 1622-1638 (2012).</li><li>Waters, P. 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=Serologic+diagnosis+of+NMO:+a+multicenter+comparison+of+aquaporin-4-IgG+assays.">Serologic diagnosis of NMO: a multicenter comparison of aquaporin-4-IgG assays.</a> Neurol. 78, 665-671 (2012).</li><li>McLaughlin, K. 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=Age-Dependent+B+Cell+Autoimmunity+to+a+Myelin+Surface+Antigen+in+Pediatric+Multiple+Sclerosis.">Age-Dependent B Cell Autoimmunity to a Myelin Surface Antigen in Pediatric Multiple Sclerosis.</a> J Immunol. 183, 4067-4076 (2009).</li><li>Probstel, A. K., 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=Antibodies+to+MOG+are+transient+in+childhood+acute+disseminated+encephalomyelitis.">Antibodies to MOG are transient in childhood acute disseminated encephalomyelitis.</a> Neurol. 77, 580-588 (2011).</li><li>Cho, 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=N-terminal+Region+of+Dopamine+D2+Receptor,+a+Rhodopsin+Family+GPCR,+Regulates+Proper+Integration+into+Plasma+Membrane+and+Endocytic+Routes.">N-terminal Region of Dopamine D2 Receptor, a Rhodopsin Family GPCR, Regulates Proper Integration into Plasma Membrane and Endocytic Routes.</a> British journal of pharmacology. , (2011).</li><li>Zhou, 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=Identification+of+a+pathogenic+antibody+response+to+native+myelin+oligodendrocyte+glycoprotein+in+multiple+sclerosis.">Identification of a pathogenic antibody response to native myelin oligodendrocyte glycoprotein in multiple sclerosis.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 19057-19062 (2006).</li><li>Brilot, F., 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=Antibodies+to+native+myelin+oligodendrocyte+glycoprotein+in+children+with+inflammatory+demyelinating+central+nervous+system+disease.">Antibodies to native myelin oligodendrocyte glycoprotein in children with inflammatory demyelinating central nervous system disease.</a> Ann Neurol. 66, 833-842 (2009).</li><li>Boronat, 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=Encephalitis+and+Antibodies+to+Dipeptidyl-Peptidase-Like+Protein-6,+a+Subunit+of+Kv4.2.">Encephalitis and Antibodies to Dipeptidyl-Peptidase-Like Protein-6, a Subunit of Kv4.2.</a> Potassium Channels. Ann Neurol. 73, 120-128 (2013).</li><li>Hinson, S. 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=Aquaporin-4-binding+autoantibodies+in+patients+with+neuromyelitis+optica+impair+glutamate+transport+by+down-regulating+EAAT2.">Aquaporin-4-binding autoantibodies in patients with neuromyelitis optica impair glutamate transport by down-regulating EAAT2.</a> J. Exp. Med. 205, 2473-2481 (2008).</li></ol>

本文介绍了一种新的应用流式细胞仪活细胞为基础的检测，以检测针对特定的细胞表面蛋白神经元使用高通量采样抗体。使用这种技术，我们报告说，受影响的自身免疫性中枢神经系统疾病的患者亚组有血清抗体表面NMDA受体或地表D2R。 使用这种高通量流式细胞仪活细胞为基础的检测为最佳的抗体检测的基本步骤包括：1）获得高产量的健康转染的细胞，2）验证高细胞表面所?...

近年来，中枢神经系统（CNS）疾病的自身免疫性形式也已确定。它已经表明，这些疾病相关联，并且由自身抗体的存在来定义。这些抗体结合到神经元的受体或参与神经传递1,2突触蛋白。不同的抗原已被检测到，如N-甲基-D-天冬氨酸受体（NMDAR）3-5，γ-氨基丁酸B受体（GABA B）受体6，α-氨基-3 -羟基-5 -甲基-4 -异唑丙酸（AMPA）受体7，电压-门控钾通道（VGKC）相关的蛋白质：富含亮氨酸胶质瘤活化蛋白1（LGL-1）和contactin相关蛋白2（capsr2）8,9，谷氨酸受体5,10和多巴胺-2受体（D2R）11。在过去，这些自身免疫CNS障碍（主要是所谓的脑炎）往往确诊和治疗。这些新型抗体的生物标志物， 如：NMDA受体抗体或抗体D2R，大幅提高了诊断和认识，并已打开患者的治疗方案。事实上，随着免疫治疗早期治疗与改善预后11,12关联。 传统的方法来检测抗体，如酶联免疫吸附测定（ELISA）和免疫印迹实验已用于检测血清中的抗体。然而，他们不容易使识别的细胞外或表面抗原决定簇和，相反，可能揭示免疫反应对细胞内抗原决定簇。此外，结合到所涉及的神经传递相关的重要蛋白质的胞外域的抗体很可能是致病2,3,7,13,14。因此，准确和灵敏的分析方法的发展，以发现相关的细胞表面或细胞外的患者抗​​体的目标是至关重要的。在该领域的金标准方法是基于使用活细胞，在所谓的“细胞巴sed的分析“。此方法涉及一种抗原的表达在哺乳动物细胞（最经常的人胚肾293（HEK293）细胞）中的天然形式由含有感兴趣抗原的完整cDNA序列的载体转染的表面上。然后活nonpermeabilized细胞与稀释的患者血清和随后的荧光染料标记的抗人免疫球蛋白（Ig）的第二抗体。荧光的强度或电平然后检测并关联到自身抗体结合的程度。这种技术是特定的，因为只有一种抗原的过度表达在细胞中。最广泛使用的“读出”已经经过免疫4,5,8-10共聚焦显微镜分析。然而，流式细胞术基于细胞的测定法已成功地用于检测抗体的患者的脱髓鞘疾病15-17。特别是，水等 15相比抗体的检测用锅的抗体的检测技术，包括基于细胞的测定接着显微镜或流式细胞仪EL分析，并且已经示出流式细胞仪的基于细胞的测定法是最灵敏，准确，可靠的方法。因此，流式细胞术基于细胞的测定是有利的，因为它是定量的，不研究者依赖性的，并且不涉及任何利用放射性物质。这也是方便，因为它允许较大的患者群在很短的时间量来测定。 最近，我们已进行了优化的高通量流式细胞术基于细胞的测定，以检测NMDA受体抗体和D2R抗体在自身免疫性疾病，中枢神经系统11。我们小组最近利用流式细胞仪细胞分析检测NMDA受体抗体在病人血清。这些NMDA受体抗体阳性血清，使用共聚焦显微镜分析的先前和也被发现是阳性11。这个协议概括了流式细胞术基于细胞的测定为C的检测onformation敏感的CNS抗体在患者血清中，利用一个自动化的高通量采样器。

<table border="0" cellpadding="0" cellspacing="0" width="751">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">pIRES2-EGFP vector</td>
			<td style="width:187px;">Clontech</td>
			<td style="width:136px;">6029-1</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Human HA-Dopamine-2 receptor (D2R) cDNA</td>
			<td style="width:187px;">Missouri S&#38;T cDNA Resource Centre</td>
			<td style="width:136px;">DRD020TN00</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Human subunit 1 of NMDAR (NR1) cDNA</td>
			<td style="width:187px;"></td>
			<td style="width:136px;"></td>
			<td style="width:173px;">Gift from Prof A. Vincent&#160; (Oxford, UK)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Monoclonal purified mouse anti-human NR1 antibody (clone: 54.1)</td>
			<td style="width:187px;">BD Pharmingen</td>
			<td style="width:136px;">556308</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Mouse purified monoclonal anti-DRD2 IgG (clone: 1B11)</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:136px;">WH0001813M1-50UG</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Alexa Fluor 647 donkey anti-mouse IgG (H + L)</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">A31571</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Alexa Fluor 647 goat anti-human IgG (H + L)</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">A21445</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:255px;">Dulbecco&#8217;s Modified Eagle Medium (1x) 4.5 g/L D-glucose, L-Glutamine and 110 mg/L sodium pyruvate</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">11995-065</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Foetal bovine serum</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">10099-141</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Gentamicin</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">15710-072</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">GlutaMAX</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">35050-061</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Penicillin-streptomycin</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">15140-122</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Geneticin</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">10131-035</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:255px;">Dulbecco&#39;s Phosphate-Buffered Saline (1x) (-Ca2+/-Mg2+)</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">14190-144</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">TrypLE Express (1x) with Phenol Red</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">12605-028</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">0.9% Sodium chloride</td>
			<td style="width:187px;">Baxter</td>
			<td style="width:136px;">AHF7975</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Polyethylenimine</td>
			<td style="width:187px;">Polysciences Inc</td>
			<td style="width:136px;">9002-98-6</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Versene</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">15040-066</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">XhoI</td>
			<td style="width:187px;">Roche</td>
			<td style="width:136px;">10899194001</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">NheI</td>
			<td style="width:187px;">Roche</td>
			<td style="width:136px;">10885843001</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">PureLink PCR Purification Kit</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">K3100-01</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">JetQuick Gel Extraction Spin Kit</td>
			<td style="width:187px;">Genomed</td>
			<td style="width:136px;">420050</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">T4 DNA Ligase</td>
			<td style="width:187px;">Roche</td>
			<td style="width:136px;">10481220001</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">Plasmid&#160;Plus&#160;Maxi Kit</td>
			<td style="width:187px;">Qiagen</td>
			<td style="width:136px;">12963</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;"></td>
			<td style="width:187px;"></td>
			<td style="width:136px;"></td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">Name of Equipment/Software</td>
			<td style="width:187px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td style="width:173px;">Model/Version</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Flow cytometer with high-throughput sampler system</td>
			<td style="width:187px;">BD Biosciences</td>
			<td style="width:136px;"></td>
			<td style="width:173px;">BDLSRII with HTS</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:255px;">Inverted microscope</td>
			<td style="width:187px;">Olympus</td>
			<td style="width:136px;"></td>
			<td style="width:173px;">CKX41 (with mercury lamp and U-RFLT50 power supply)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Electronic multichannel pipette (15-300 &#956;l)</td>
			<td style="width:187px;">Eppendorf</td>
			<td style="width:136px;">613-2240P</td>
			<td style="width:173px;">12-channel Xplorer Plus</td>
		</tr>
		<tr height="56">
			<td height="56" style="height:56px;width:255px;">Excel</td>
			<td style="width:187px;">Microsoft</td>
			<td style="width:136px;"></td>
			<td style="width:173px;">2010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Prism</td>
			<td style="width:187px;">GraphPad Software, Inc.</td>
			<td style="width:136px;"></td>
			<td style="width:173px;">v4</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:255px;">FlowJo</td>
			<td style="width:187px;">Treestar</td>
			<td style="width:136px;"></td>
			<td style="width:173px;">v7.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">50 ml polypropylene conical tubes</td>
			<td style="width:187px;">BD Biosciences</td>
			<td style="width:136px;">352070</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">6-well plate</td>
			<td style="width:187px;">BD Biosciences</td>
			<td style="width:136px;">353046</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tissue culture flask (T75)</td>
			<td style="width:187px;">BD Biosciences</td>
			<td style="width:136px;">353136</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Parafilm</td>
			<td style="width:187px;">Pechiney Plastic Packaging</td>
			<td style="width:136px;">PM-992</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">V-bottom 96-well plate</td>
			<td style="width:187px;">Corning</td>
			<td style="width:136px;">651180</td>
			<td style="width:173px;"></td>
		</tr>
	</tbody>
</table>

high-throughput flow cytometry cell-based assay to detect antibodies to n-methyl-d-aspartate receptor or dopamine-2 receptor in human serum

在最近几年，抗表面和参与神经传递构象的蛋白已在儿童和成人自体免疫CNS疾病检测。这些抗体已被用于指导诊断和治疗。基于细胞的测定具有改善的抗体在患者血清中的检测。它们是基于大脑的抗原上的真核细胞中，然后将其用稀病人血清随后荧光标记的二抗温育的表面表达。洗涤后，将二级抗体结合通过流式细胞术，然后进行分析。我们的小组已经开发出一种高通量流式细胞仪活细胞为基础的检测可靠地检测抗体的特异性神经递质受体。这种流式细胞仪的方法是直接的，定量的，高效的，并且使用高通量采样系统允许大量患者组可以很容易地测定在很短的时间空间。此外，这种细胞系作为说可以容易地适用于检测抗体，以许多不同的抗原靶，无论是从中枢神经系统和外周。发现更多的新型抗体的生物标志物，使迅速和准确的诊断和提高治疗免疫介导的疾病。

购入住HEK293 D2R +和HEK293细胞的CTL在流式细胞仪中使用的高通量采样器。在分析过程中，细胞是基于前向散射（大小）和侧向散射光（粒度）参数（ 图1A和1D）门控。转染的HEK293细胞中表达的报道分子，绿色荧光蛋白，在细胞质中，与未转染的细胞被排除在分析（ 图1B和1E）。内的GFP +栅极，与AF647-标记的抗人IgG第二抗体结?...

Watch this Scientific Journal Video about High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum at JoVE.com

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

在最近几年，活细胞检测已被成功地用于检测抗体的表面和构象的抗原。在这里，我们利用高通量流式细胞仪使的患者大样本的分析描述的方法。检测新抗体将提高诊断的免疫介导的病症和治疗。

高通量流式细胞仪检测细胞为基础的试验检测抗体，以N-甲基-D-天冬氨酸受体或多巴胺2受体在人血清

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Research

JoVE Journal

Medicine

The Measurement and Treatment of Suppression in Amblyopia

测量和制止在弱视治疗

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

科研

医学

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

白细胞亚群在暂时或永久的脑缺血模型体视学和流式细胞仪鉴定

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism (Microcystis aeruginosa) Using Flow Cytometry

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism Microcystis aeruginosa Using Flow Cytometry

分子探针的优化，以确定细胞死亡的光合生物（<em&gt;铜绿微囊藻</em&gt;）使用流式细胞仪

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. ...

Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

乳腺癌代谢调控的系统生物学雌激素受体信号

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective ...

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status

高通量，复用和有针对性的蛋白质组学分析脑脊液量化神经退行性生物标志物和载脂蛋白E亚型状态

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be ...

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

一种基于动力学荧光的 Ca2 +动员法, 用于识别 G 蛋白偶联受体激动剂、拮抗器和构调制器

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

应用流式细胞仪对人体脂肪组织免疫细胞的表征

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

高通量硝基苯并二氮唑标记的胆固醇积液测定

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in ...

Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E (sIgE)

Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E sIgE

生物芯片微流控技术在血清过敏源特异性免疫球蛋白 E (sIgE) 检测中的应用

Allergic disease is common in both adults and children. Identification of the causative allergens is significant in disease management and prevention. ...

Improved Renal Denervation Mitigated Hypertension Induced by Angiotensin II Infusion

改善肾去神经支配缓解血管紧张素II.诱导的高血压

The benefits of renal sympathetic denervation (RDN) on blood pressure have been proved in a large number of clinical trials in recent years. However, the ...