Name: sensitive detection of proteopathic seeding activity with fret flow cytometry
Uploaded: 2015-12-08T13:00:00
Description: Watch this Scientific Journal Video about Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry at JoVE.com

This work was supported by the Tau Consortium (M.I.D); National Institutes of Health Grant 1R01NS071835 (M.I.D.), a Department of Defense Grant PT110816 (to M.I.D.), 1F32NS087805 (to J.L.F.), and 1F31NS079039 (to B.B.H.).

注：该协议强调利用FRET的流式细胞仪从小鼠生物样本检测播种活动。它还与重组原纤和人类生物样品兼容。按照IACUC批准的程序进行鼠标安乐死与脑收获。 1.脑提取<ol><li>以下用异氟烷（2％）深麻醉，灌注的小鼠用含有0.03％肝素冰冷的PBS，并提取在以下量具等描述的细节的大脑 。18 </li><li>放置提取的组织在一个冷冻小瓶中，并通过将在液氮中冷冻单元。可选择地，?...

<ol>
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此处所描述的FRET流式细胞仪系统是一个功能强大的工具，快速和定量地评估tau蛋白播种活性。它只需要适度的细胞培养的经验和FRET的应用知识和流式细胞仪。其他播种测定法，如硫磺素T - 这表现出增强的荧光当结合β片层结构 - 是费力的，需要一个纯的，重组蛋白质底物。此外， 在体外播种测定法的tau仅半定量的并且通常不敏感种子材料23,24的亚纳摩尔水平。 FRET流式细胞仪，但...

细胞内的tau淀粉样蛋白的积累定义τ病变如阿尔茨海默氏病。在早期疾病阶段，病理通常定位于大脑的离散区域，但随着疾病进展，病理学总是沿不同的神经网络1-5传播。越来越多的证据表明，有毒蛋白聚集体跨细胞繁殖underlies此病理（6-10综述）。在这个模型中，proteopathic种子（例如，tau蛋白）释放从供体细胞，并进入邻近细胞，转化天然tau蛋白成经由模板化的构象变化11-15错误折叠形式。此处所描述的测定法的开发灵敏检测这种播种活性。它是用重组蛋白和生物样品相容并允许proteopathic播种活动16分钟程度的定量。 HEK 293T细胞稳定表达tau蛋白重复ðomain（RD）含有与疾病相关的突变P301S融合到CFP或YFP（以下简称为tau蛋白-RD-CFP / YFP的细胞）作为播种活性的稳定的生物传感器。在没有proteopathic种子，细胞保持tau蛋白作为水溶性单体，和具有没有明显的背景的FRET。自然吸收或牛头种子脂质体介导转导到细胞内，但是，结果在RD-CFP和RD-YFP聚集，产生的是单个细胞内通过流式细胞仪测量的FRET信号。 该测定的众多成分设计，以提高灵敏度和降低可变性。带1的单克隆细胞系：1的RD-CFP / YFP表达比率被选择的，因为它提供了最佳的信号：噪声。为了增加灵敏度，磷脂用来引入种子直接导入细胞（尽管研究摄取生物学机制，这可以省略）。最后，流式细胞仪监测FRET在群体水平和单个细胞<html>平，与其他蛋白质聚集测定。最终的测量结果，集成的FRET密度，是高度的定量和两对细胞凝集的数量，和聚集已经发生向其中每个单元内的程度帐户。所有这些优化的参数，提高了灵敏度，并确保重现性。 该系统最近采用了全面的研究转基因P301S tau蛋白病小鼠17进行评估的时间开始和相关头播种活动进展到其他常用的tau病理学标志（ 例如 ，MC1，AT8，PG5，并ThioflavinS）。播种活动是迄今为止头病理的最早和最强大的标志物进行评估，组织学检测由至少6周之前。播种活性似乎在1.5个月，并逐渐随着年龄增大，这表明proteopathic种子在其发作和/或神经变性16的级数的因果作用。 e_content“&gt;从生物样品中的种子材料分钟水平的精确定量可以促进监测早期疾病进展的研究。通过缩短测试时间并且使得能够使用较年轻的动物，这可能增加的临床前动物试验的效率和准确性。例如，在在P301S鼠标如前所述，铅化物可作为交付早在4-6周，2-4周后进行疗效监测。该法应准确量化播种的任何削减（，或在发病播种活动的前夕）活性。FRET流式细胞仪已经在体外筛选的应用，以及，例如，抗tau蛋白的试剂（例如，抗体，小分子等），可以迅速地对它们阻断直接在培养接种诱导，使用任一重组tau蛋白的能力进行测试聚集体或脑源性裂解物作为种子源（图5）。采用这种设置，一旦晶种材料制备，一个前periment只需要三天完成，包括数据分析。 proteopathic播种活动的快速定量因此可以促进神经退行性疾病的许多研究。

<table>
	<tbody>
		<tr>
			<td>TBS</td>
			<td>Sigma</td>
			<td>T5912</td>
		</tr>
		<tr>
			<td>cOmplete Protease Inhibitors (EDTA-free)</td>
			<td>Roche</td>
			<td>4693159001</td>
		</tr>
		<tr>
			<td>Cryo-vials</td>
			<td>Sarstedt</td>
			<td>72.694.006</td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>Mettler Toledo</td>
			<td>XSE 105DU</td>
		</tr>
		<tr>
			<td>Weighing Boats</td>
			<td>Fisher Scientific</td>
			<td>13-735-743</td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>USA Scientific</td>
			<td>1475-0501</td>
		</tr>
		<tr>
			<td>Omni Sonic Ruptor Ultrasonic Homogenizer</td>
			<td>Omni International</td>
			<td>18-000-115</td>
		</tr>
		<tr>
			<td>Micro-Tip for Ultrasonic Homogenizer</td>
			<td>Omni International</td>
			<td>OR-T-156</td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Fisher Scientific</td>
			<td>A451</td>
		</tr>
		<tr>
			<td>Noise Cancelling Ear Muffs</td>
			<td>Fisher Scientific</td>
			<td>19-145-412</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Fisher Scientific</td>
			<td>S47299</td>
		</tr>
		<tr>
			<td>1.5 mL tubes</td>
			<td>USA Scientific</td>
			<td><a href="http://www.usascientific.com/Seal-Rite-1.5-ml-tube-sterile.aspx">1615-5510</a></td>
		</tr>
		<tr>
			<td>Microcentrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>5424 000.215</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Life Technologies</td>
			<td>14190-136</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11965-084</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>HyClone</td>
			<td>SH30071.03</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Life Technologies</td>
			<td>35050-061</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
		</tr>
		<tr>
			<td>50 mL Conical Tubes</td>
			<td>Phenix Research</td>
			<td>SS-PH15</td>
		</tr>
		<tr>
			<td>25 mL reagent resevoirs</td>
			<td>VWR</td>
			<td>41428-954</td>
		</tr>
		<tr>
			<td>Multi channel pipet</td>
			<td>Fisher Scientific</td>
			<td>TI13-690-049</td>
		</tr>
		<tr>
			<td>96 well flat bottom plates</td>
			<td>Corning</td>
			<td>3603</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668019</td>
		</tr>
		<tr>
			<td>96 well round bottom plates</td>
			<td>Corning</td>
			<td>3788</td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>RT 15710</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>P5493</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>ED2SS</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Life Technologies</td>
			<td>14185-052</td>
		</tr>
		<tr>
			<td>Sorvall ST 40 Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004509</td>
		</tr>
		<tr>
			<td>BIOLiner Swinging Bucket Rotor</td>
			<td>Thermo Scientific</td>
			<td>75003796</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>VWR</td>
			<td>15170-172</td>
		</tr>
		<tr>
			<td>MACSQuant VYB Flow Cytomter</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-116</td>
		</tr>
		<tr>
			<td>Chill 96 Rack</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-459</td>
		</tr>
		<tr>
			<td>Flow Jo analysis software</td>
			<td>Flow Jo</td>
			<td></td>
		</tr>
		<tr>
			<td>20 uL pipet tips</td>
			<td>Rainin</td>
			<td>GPS-L10</td>
		</tr>
		<tr>
			<td>200 uL pipet tips</td>
			<td>Rainin</td>
			<td>GPS-250</td>
		</tr>
		<tr>
			<td>1 mL pipet tips</td>
			<td>Rainin</td>
			<td>GPS-1000</td>
		</tr>
		<tr>
			<td>200 uL pipet tips</td>
			<td>USA Scientific</td>
			<td>1111-1800</td>
		</tr>
		<tr>
			<td>5 mL serological pipett</td>
			<td>Phenix Research</td>
			<td>SPG-606180</td>
		</tr>
		<tr>
			<td>10 mL serological pipett</td>
			<td>Phenix Research</td>
			<td>SPG-607180</td>
		</tr>
		<tr>
			<td>25 mL Serological pipett</td>
			<td>Phenix Research</td>
			<td>SPG-760180</td>
		</tr>
	</tbody>
</table>

sensitive detection of proteopathic seeding activity with fret flow cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

FRET流式细胞仪能敏感，定量，并从重组或生物样本快速检测播种活动。实验设置是容易的：表达的tau-RD-CFP / YFP单克隆来源的稳定细胞系转导的种子材料，孵育24-48小时，并进行流式细胞术分析（图1A）。在没有种子，生物传感器细胞保持tau蛋白以可溶，单体形式（图1B）。在种子的情况下，然而，生物传感器细胞将tau蛋白为凝聚状态（图1C）中，产生一个是?...

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Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

与FRET流式细胞仪灵敏地检测Proteopathic播种活动

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

Research

JoVE Journal

Bioengineering

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

由多光谱成像流式细胞仪分析细胞内化纳米粒子和细菌

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

科研

生物工程

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

一个手机上的宽视场荧光显微镜和荧光成像流式细胞仪

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general ...

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

Flying Insect Detection and Classification with Inexpensive Sensors

飞行昆虫检测与分类用便宜的传感器

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow ...

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

蛋白质的MS检测快速酶法处理与流通型微反应器

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

通过流式细胞术分离原代小鼠视网膜神经节细胞（RGC）

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

使用多光谱成像流细胞测定法执行体外微核测定的自动化方法

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Ovarian Cancer Detection Using Photoacoustic Flow Cytometry

使用光声流细胞测定卵巢癌检测

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies ...