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（例如的NheI和XhoI人类D2R的cDNA）。 </li><li>受限制的pIRES2-EGFP载体内结扎切断的cDNA插入。 </li><li>序列连接的pIRES2-EGFP D2R或NMDA受体载体来检查向量是否包含正确的顺序。 </li><li>净化和放大的pIRES2-EGFP D2R或NMDA受体载体转染后用。 </li></ol></li></ol>书名“&gt; 2。HEK293细胞转染的神经元抗原的表达<ol><li>培养HEK293细胞在组织培养瓶中用新鲜的Dulbecco改良的Eagle培养基（1X）（DMEM）中完全用4.5 g / L的D-葡萄糖，L-谷氨酰胺和110mg / L丙酮酸钠补加有10％胎牛血清（FBS）， 2毫米的GlutaMAX，和50微克/毫升庆大霉素。 </li><li>用胰蛋白酶和转移到锥形管分离HEK293细胞。 <ol><li>通过在250×g离心6分钟离心细胞洗涤并重新悬浮在新鲜的完全DMEM培养液将细胞沉淀。 </li></ol></li><li>计数细胞和种子6孔板中在大约8×10 5个细胞/孔，并培养过夜2毫升的DMEM，在37℃和5％CO 2的培养箱中。 </li><li>一旦它们达到约70％汇合时，转染的HEK293细胞与质粒pIRES2-EGFP NR1（HEK293 NMDAR +细胞），质粒pIRES2-EGFP D2R（HEK293 D2R +细胞）和质粒pIRES2-EGFP（HEK293 CTL细胞;矢量控制），如下所示。稍后继续对一些未转染的HEK293细胞进行赔偿。 <ol><li>制备转染混合物，其包含2.5微克的DNA中加入200μl0.9％的氯化钠和4微克/毫升的聚乙烯亚胺/孔（每含2ml新鲜的完全DMEM）中的所需的体积。 </li><li>立即旋涡振荡混合10秒和孵化在室温（RT）10分钟，加入转染混合物的体积，以适当的井前。 </li><li>覆盖板，敷用封口膜的两侧，并离心分离，在280×g离心5分钟，以帮助细胞转染。 </li><li>除去封口膜，然后在孵化器的地方文化在37°C </li><li> 18小时后用新鲜完全DMEM更换培养基，并在文化保持另外72小时。 </li><li>可选：72小时转染后，转染的细胞可以通过培养在完全DMEM补充有250微克/毫升遗​​传霉素选择的，以便获得多克隆稳定转染。 </li></ol></li></ol><p类=“jove_title”&gt; 3。流式细胞仪检测细胞为基础的检测方法抗体的神经元表面抗原<ol><li>分离HEK293 NMDAR +，HEK293 D2R +，和HEK293细胞的CTL孵育，用500μL/孔的Versene约5分钟，在37℃下<ol><li>重悬在2ml/well的PBS（ -的Ca 2 + /-Mg 2 +的）补充有2％FBS（PBS / FBS）中，并转移到15毫升或50毫升的锥形试管中。 </li><li>通过离心分离，在250×g离心6分钟洗涤细胞，重悬细胞沉淀在15-50毫升PBS / FBS中。重复洗2倍。 </li><li>计数细胞，然后悬浮在PBS / FBS的以1×10 6细胞/ ml。 </li></ol></li><li>设计的96孔模板进行流式细胞术获取，考虑到HEK293 D2R +或HEK293 NMDAR +细胞，以及HEK293细胞的CTL将必须与每个患者样本或初级抗体一起温育。 </li><li>种子细胞在V形底96孔板中以50,000细胞/我们LL根据流式细胞仪采集模板的流动。 </li><li>通过离心96孔板中，在450×g离心5分钟，并轻轻地用电子或手动多道移液器除去上清液，于一角度倾斜板，并小心不要吸出细胞沉淀沉淀细胞。 </li><li>培养HEK293 D2R +，HEK293 NMDAR +，和HEK293细胞的CTL 1小时在室温下与黑暗： <ol><li>第一抗体，以评估抗原的表面表达的系列稀释。 </li><li>为了检测抗体的患者样品。 </li></ol></li><li>使用适当的流式细胞仪检测补偿控制， 例如 。未染色的未转染的HEK293细胞中，GFP + HEK293细胞（AF647 - ）和未转染的AF647 + HEK293细胞（GFP）。 </li><li>通过离心分离，在450×g离心5分钟，洗涤细胞，重悬细胞沉淀在170微升PBS / FBS中。重复洗涤步骤两次以上。 </li><li>孵育细胞1小时R最快室温在黑暗中以1:100稀释AF647-标记的二抗（抗人IgG对患者血清和抗小鼠IgG抗-NR1或抗D2R初级抗体）。 </li><li>洗涤细胞三次，如在步骤3.7，并重新悬浮于35微升PBS / FBS的流式细胞术细胞采集。 </li><li>设置在流式细胞仪（BDLSRII）流动的高通量采样器（HTS）。 </li><li>获得10,000个事件/孔根据流式细胞仪采集模板的流程。 </li><li>导出，然后分析数据，使用流式细胞仪软件包，如FlowJo V7.5分析： <ol><li>首先，基于前向（大小）和侧面（粒度）门活HEK293细胞分散。 </li><li>基于高GFP-阳性分析仅转染的细胞进一步栅极活的HEK293细胞。 </li><li>确定活GFP阳性细胞内的AF647信道的平均荧光强度（MFI）。 </li><li>数据导出到Excel或棱镜进行分析： <ol><li> C区初级抗体（抗NMDA受体或抗D2R抗体）相对于MFI的oncentrations从孵育这些抗体的细胞获得。 </li><li>对于每一个病人和对照样品，减去从HEK293 D2R +或NMDA受体的HEK293 +细胞，小额信贷机构分别HEK293 CTL细胞的MFI确定ΔMFI。 </li><li>通过将上述控制队列的平均ΔMFI三个标准差计算抗体阳性的门槛。 </li><li>情节个别样品根据其血清类型分组上的曲线图，并表示所述阈值作为线。 </li></ol></li></ol></li></ol>

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利益冲突：一个专利已提交FB和RCD（悉尼大学）声称D2R为目标的自身抗体。

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。

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			<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>
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		</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 ...

high-throughput-flow-cytometry-cell-based-assay-to-detect-antibodies

Research

JoVE Journal

Medicine

16.1K 次观看.  The University of Sydney. 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。 

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

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

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

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

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 ...

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e., high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.

This study successfully adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research.

适应人类Videofluoroscopic燕子研究的方法来检测和检定吞咽困难的小鼠疾病模型

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

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

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective mining of the information in the large-scale datasets that are obtained from gene expression microarrays, deep sequencing experiments or metabolic profiling is essential to uncover and then effectively target the critical regulators of diseased cell phenotypes. Estrogen Receptor &#945; (ER&#945;) is one of the master transcription factors regulating the gene programs that are important for estrogen responsive breast cancers. In order to understand to role of ER&#945; signaling in breast cancer metabolism we utilized transcriptomic, cistromic and metabolomic data from MCF-7 cells treated with estradiol. In this report we described generation of samples for RNA-Seq, ChIP-Seq and metabolomics experiments and the integrative computational analysis of the obtained data. This approach is useful in delineating novel molecular mechanisms and gene regulatory circuits that are regulated by a particular transcription factor which impacts metabolism of normal or diseased cells.

Integration of data from genome-wide sequencing experiments and metabolomics experiments is a challenge. In this paper we report, for the first time, generation, analysis and integration of transcriptome, cistrome and metabolome data from breast cancer cells treated with estradiol.

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

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be important in the development of future novel therapies. Often&#160;potential new biomarkers do not make it&#160;into the clinic&#160;due to limitations&#160;in their development and&#160;high costs.&#160;However,&#160;targeted proteomics using Multiple Reaction Monitoring Liquid Chromatography-tandem/Mass Spectrometry (MRM LC-MS/MS), specifically using triple quadrupole mass spectrometers, is one method that can be used to rapidly evaluate and validate biomarkers&#160;for clinical translation into diagnostic laboratories. Traditionally, this platform has been used extensively for measurement of small molecules&#160;in clinical laboratories, but it is the potential to analyze proteins, that makes it an attractive alternative to ELISA (Enzyme-Linked Immunosorbent Assay)-based methods. We describe here how targeted proteomics can be used to measure multiplexed markers of dementia, including the detection and quantitation of the known risk factor apolipoprotein E isoform 4 (ApoE4).
In order to make the assay suitable for translation, it is designed to be rapid, simple, highly specific and cost effective. To achieve this, every step in the development of the assay must be optimized for the individual proteins and tissues they are analyzed in. This method describes a typical workflow including various tips and tricks to developing a targeted proteomics MRM LC-MS/MS for translation.
The method development is optimized using custom synthesized versions of tryptic quantotypic peptides, which calibrate&#160;the MS for detection and then spiked into CSF to determine correct identification of the endogenous peptide in the chromatographic separation prior to analysis in the MS. To achieve absolute quantitation, stable isotope-labeled internal standard versions of the peptides with short amino acid sequence tags and containing a trypsin cleavage site, are included in the assay.

We describe a high-throughput, multiplex, and targeted proteomic cerebrospinal fluid (CSF) assay developed with potential for clinical translation. The test can quantitate potential markers and risk factors for neurodegeneration, such as the apolipoprotein E variants (E2, E3 and E4), and measure their allelic expression.

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

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include well-validated targets for therapeutic intervention. Discovery of lead compounds, including small synthetic molecules, that specifically inhibit the receptor&#39;s function, is an important initial step in drug development and relies on sensitive, specific, and robust cell-based assays. Here, we describe a kinetic cellular assay with a fluorescent readout primarily designed to identify receptor-specific antagonists that inhibit the intracellular Ca2+ release evoked upon the activation of the CXC chemokine receptor 4 (CXCR4) by its endogenous ligand, the CXC chemokine ligand 12 (CXCL12). A key advantage of this method is that it also enables screening of compounds endowed with intrinsic agonistic properties (i.e., compounds eliciting an increase in intracellular Ca2+ concentration in the absence of CXCL12) or compounds modulating the receptor&#39;s function via interaction with allosteric binding sites (i.e., positive and negative allosteric modulators (PAMs and NAMs, respectively)). On the down side, autofluorescent compounds might interfere with the assay&#39;s readout, thereby hampering reliable data interpretation. Most likely this assay can be implemented, with minimal adaptations, as a generic drug discovery assay for many other GPCRs of which the activation leads to a release of intracellular Ca2+.

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

The described cellular assay is designed for the identification of CXC chemokine receptor 4 (CXCR4)-interacting agents that inhibit or stimulate, either competitively or allosterically, the intracellular Ca2+ release initiated by CXCR4 activation.

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

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis 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

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

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)

Allergic disease is common in both adults and children. Identification of the causative allergens is significant in disease management and prevention. However, a specific immunoglobulin E (IgE) measurement system with a high price-performance ratio is lacking in mainland China, especially in the primary care hospitals. This paper describes the principle and operation procedures of using a microfluidic cartridge-based chemiluminescence system to detect allergen-specific IgE in serum. The results were compared with those from ImmunoCAP (System 1), the industrial standard, and the reproducibility of the system to detect patients sensitized to common allergens is evaluated. The results showed that in comparison with ImmunoCAP (System 1), the BioIC System (System 2) has good precision and sensitivity in detecting serum-specific IgE against various inhalant and food allergens but with a significantly lower cost. It can serve as a good alternative to System 1 in primary care hospitals in mainland China who have lower financial affordability.

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

This paper presents a protocol for detecting serum-specific immunoglobulin E with a microfluidic cartridge-based chemiluminescence system and the evaluation of its usefulness in allergy diagnoses.

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. ...

大鼠膝骨关节炎的标准化推拿手法

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its mechanism. Therefore, the manipulation of animal models of knee osteoarthritis is critical. This protocol provides a standard process for Tuina manipulation on KOA rats and a preliminary exploration of the mechanism of Tuina for KOA. The press and kneading manipulation method (a kind of Tuina manipulation that refers to pressing and kneading the specific area of the body surface) is applied on 5 acupoints around the knee joint of rats. The force and frequency of the manipulation were standardized by finger pressure recordings, and the position of the rat during manipulation is described in detail in the protocol. The effect of manipulation can be measured by pain behavior tests and microscopic findings in synovial and cartilage. KOA rats showed significant improvement in pain behavior. The synovial tissue inflammatory infiltration was reduced in the Tuina group, and the expression of tumor necrosis factor (TNF)-&#945; was significantly lower. Compared to the control group, chondrocyte apoptosis was less in the Tuina group. This study provides a standardized protocol for Tuina manipulation on KOA rats and preliminary proof that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis.

作者聚焦：了解大鼠推拿操作的机制 

We present a protocol for Tuina manipulation performed on plaster-immobilized-induced knee osteoarthritis rats. Based on the preliminary results, it suggested that the efficacy of the method relied on the reduction of inflammation and cartilage loss.

推拿操作以减少膝关节骨关节炎大鼠的炎症和软骨损失

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its ...