Name: protein digestion, ultrafiltration, and size exclusion chromatography to optimize the isolation of exosomes from human blood plasma and serum
Uploaded: 2018-04-13T15:00:00.000+00:00
Duration: 9 min 22 s
Description: Watch this Scientific Journal Video about Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum at JoVE.com

这项研究得到了来自 CSU (KMD)、ATCC 合同 #2016 0550-0002 (NIAID HHSN272201600013C 分包)、比尔和梅琳达盖茨基金会 (OPP1039688) (KMD/NKG) 的内部资金的支持。我们感谢美国国家科学基金会在科罗拉多州立大学 (拉吴) 暑期项目的研究经验, 以得到更多的支持。

这项工作在科罗拉多州立大学的机构审查委员会 (IRB) 初步审查后被确定为非人类专题研究, 因为所有的人类样本都是从 Bioreclamation IVT biorepository 中提取出来的样本, 并根据 IRB 批准的协议收集。
1. 通过造粒较大的囊泡和细胞碎片制备粗样品 (血浆或血清)
注意:安全考虑: 血浆、血清和其他生物液体是 biohazardous 材料, 必须特别小心处理, 同时佩戴基本的个人防护设备, 包括手套和实验室大衣。强烈建议生物样品在生物安全柜中处理;如果不可用, 建议使用眼睛保护。
<ol><li>平衡在4摄氏度冰箱 (从-20 或-80 °c 贮存) 中, 将管置于非震动条件下, 将血清或血浆样品放在一夜之间。</li>
	<li>整除250µL 的样品进入离心管, 使用1000µL 吸管。</li>
	<li>离心样品在 1.8万 x g 30 分钟在4°c。</li>
	<li>使用200µL 吸管, 移除200µL 的清除上清液, 并将其添加到新的离心管。在转移上清时避免任何颗粒物质。丢弃颗粒材料。</li>
	<li>使用制造商的协议, 定量测定样品中的蛋白质含量 (二辛可宁酸)。要进行化验, 稀释1:50 在1x 磷酸盐缓冲盐水 (PBS) 样品。以重复的方法测试每个示例。 注意:在整个协议中使用 1x PBS, 除非另有说明。平均, 200 µL 的澄清血清或血浆 (1.8万 x g) 将产生15毫克的总蛋白。其他样本类型, 蛋白质含量较低或较高, 可能需要额外的稀释。</li>
	<li>整除10毫克的样品进入一个新的离心管。使用步骤1.5 中获得的浓度来计算相应的样品体积。用200µL 吸管整除样品。 注意:建议将剩余的样品保存在摄氏-80 摄氏度。整除以每管10毫克的不同管子为例, 以避免今后使用多种冻融循环。</li>
</ol>2. 非 exosomal 血蛋白的消化
注意:消化步骤旨在减少与外来体共同纯化的非 exosomal 蛋白。如果下游分析需要保存外切表面蛋白, 应考虑到这一步骤有可能干扰这种分析。exosomal CD63 的检测是一种表面暴露的 tetraspanin 蛋白, 对此过程没有显著的负面影响。
<ol><li>在 PBS 中500µg/毫升的浓度下制备蛋白酶 K 溶液。在圆锥管中加入相应数量的蛋白酶 K。然后加入缓冲和漩涡在三十年代, 3x 在室温下。</li>
	<li>添加50µL 蛋白酶 K 溶液的10毫克样品整除, 并轻轻地混合吹打上和下使用200µL 吸管。</li>
	<li>在37摄氏度的水浴中孵化样品30分钟. 将管子放在浮子管架中, 避免在水浴中完全浸入管内, 以避免潜在的渗漏。</li>
	<li>将样品和浮动管架转移到60°c 水浴中10分钟, 使蛋白酶 K 失效。</li>
</ol>3. 离心超滤去除小蛋白和多肽
<ol><li>在使用前, 用 PBS 冲洗含有 100 kDa 分子量截止 (截留分子量) 过滤器的超滤装置。通过将500µL 的 PBS 添加到过滤器中, 使用1000µL 吸管。旋转设备在 3700 x g 5 分钟丢弃任何剩余的 retentate 和洗脱液。 注意:超滤装置的样品容积容量必须为0.5 毫升-4 毫升, 以确保最终减少的样品体积为50µL 是可以实现的。</li>
	<li>从步骤2.4 中抽取样本, 并通过添加 PBS 将卷增加到500µL。将样品注入预冲洗的超滤装置。 注意:平均来说, 从步骤2.4 的样本量是185µL (120 到150µL 的样品和50µL 的蛋白酶 K 溶液)。</li>
	<li>将超滤装置放置在固定角度的台式离心机上, 在 3700 x 克4摄氏度, 直到样品减少到50µL 的体积。 警告:当使用超滤装置时, 必须注意避免过滤膜在离心过程中完全干燥。</li>
	<li>将500µL 的 PBS 直接添加到滤膜中, 吸液器上下5次。离心机和步骤3.3。执行此洗涤步骤共3次。</li>
	<li>将最终的50µL retentate 转移到新的离心管中。</li>
	<li>用200µL PBS 冲洗滤膜, 吹打上下10次, 从步骤3.5 将洗涤液转到管。</li>
	<li>将滤芯置于新管的反位置。运行反向旋转恢复5分钟在 2000 x g 检索剩余的样品从膜。将示例与步骤3.5 中的示例一起进行池。</li>
</ol>4. 粒度排除色谱法纯化囊泡 (秒)
<ol><li>添加850µL 的截留分子量浆, 有 700 kDa 的珠子, 进入一个空的, 上限的重力流柱使用1000µL 吸管。将列放置在适当的大小合适的机架上, 安装有一个滴水托盘。</li>
	<li>Uncap 柱底部, 让液体排泄5分钟。一旦排水, 添加10毫升的 PBS, 以洗涤树脂。允许20分钟的洗涤从列排出。</li>
	<li>将浓缩的蛋白酶 K 处理后的样品从步骤3.5 放入15毫升圆锥管中, 并将样品体积增加到5毫升, 用血清吸管添加 PBS。通过摇摆1分钟轻轻地混合管, 然后慢慢地将样品应用到步骤4.2 中用血清学吸管制备的柱上。 注意:一旦去掉柱帽, 样品就会通过该树脂的重力流动;5毫升的样品将完全流经列在10分钟。</li>
	<li>收集流动通过在一个新的15毫升圆锥管。</li>
	<li>使用血清吸管, 再次将收集到的材料应用于树脂, 以除去 700 kDa 以下的剩余材料。</li>
	<li>收集流动通过在一个新的15毫升圆锥管。清洗树脂两次添加1毫升 PBS。从步骤4.5 中收集管道中的流量;最终的总体积将约为7毫升。 注意:在步骤4.6 之后, 样品将被稀释大约35次从原始的样品容量;以下步骤将减少体积, 浓缩纯化的外切样品。</li>
</ol>5. 纯化外来体浓度和总蛋白量化
<ol><li>在使用前, 用 3 kDa 截留分子量过滤器冲洗一个超滤装置, 如步骤3.1 所述。</li>
	<li>从步骤4.6 到超滤装置和离心机在 3700 x g 4 摄氏度的摆动斗转子中添加样品。缓冲区将通过筛选器, 并且可以被丢弃。浓缩外来体将保留在过滤器上方。离心样品, 直到过滤器 (retentate) 上的容积减少到200µL。</li>
	<li>将 retentate 转移到新的离心管。</li>
	<li>用200µL PBS 冲洗滤膜, 吹打膜10次, 从步骤5.3 将洗涤液转移到管子上。这将允许收集任何剩余的外切样本。</li>
	<li>通过添加 PBS, 使样品体积达到500µL。通过缓慢吹打的混合, 上下10次。 注意:协议可以在这里暂停。样品应存放在4°c 隔夜或 20/-80 摄氏度, 长期储存。</li>
	<li>使用制造商的协议, 定量测定样品的蛋白质含量。稀释样品1:10 和1:50 在 PBS。重复运行每个示例。 注意:从血清或血浆总蛋白的10毫克开始, 从步骤5.6 中纯化外来体的总蛋白产量平均为150µg. 如果使用血清或血浆以外的样品, 则可能需要额外的稀释;产量可能因样品类型而异。</li>
</ol>6. 纳米粒子跟踪分析 (NTA) 对外来体的定量和上浆
<ol><li>添加5µg 的纯化外来体 (量化在 5.6) 到1毫升的 PBS 和漩涡十五年代的低中速度。<ol>
<li>将样品放入1毫升一次性注射器中。如果可用, 将注射器设置在自动注射器泵中, 以30µL/分钟的速度注入稀释的外切样品。</li>
		<li>使用视频捕获设置执行 NTA 测量: 屏幕增益为 3-4, 摄像机电平为 12-13。</li>
		<li>定义用于分析的脚本, 以便至少在三十年代使用每个复制的常量流运行三技术复制。对于分析, 请将捕获阈值设置为5。 注意:有关使用自动注射器的详细信息以及视频捕获的设置可在参考13中获得。NTA 的捕获和分析设置必须在不同的示例之间保持一致, 以确保可比性。</li>
	</ol>
</li>
</ol>7. 可溶性蛋白还原的测定
<ol><li>将纯化外来体的 1-10 µg 添加到 SDS 样品缓冲液中, 在 1x MES 中使用 4-12% 双三凝胶进行聚丙烯酰胺凝胶电泳 (页), 在 35 v. 通过应用电压 o 将解析蛋白转移到硝化棉200µm 膜上f 50 V 为 1-1.5 h. 执行西方印迹分析, 评估白蛋白, 载脂蛋白 A 和 B 的存在, 和外切标记 CD 63 遵循标准方法。 注意:7.1 中的方法的完整协议可以在引用14中找到。具体地说, SP007: 聚丙烯酰胺凝胶和 SP011 的运行: 西方的印迹协议。</li>
	<li>用页面和步骤7.1 分开10µg 纯化外来体, 用考马斯染料染色蛋白质带, 遵循标准建议, 以可视化蛋白质变化和样品之间的减少。 注意:对于特定于 CD63 的西方印迹协议, 进一步的细节由迪亚兹et描述。15
</li>
</ol>

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作者没有什么可透露的。

从血液中提高外来体纯度和产量的优化方法, 将提高准确地将细胞外囊泡作为几种疾病的生物标志物来源的能力。目前用于隔离外来体的标准方法, 特别是离心和沉淀方法, 有几个缺点, 包括外切聚合和可溶性蛋白造粒 7, 17, 18。另外, SEC 的使用也显示了外切隔离的显著改善, 保护外来体不被聚合, 并改进了清除常见的污染物非 exos...

外来体是 nanovesicles (大小从30毫微米到150毫微米) 释放的几乎所有的细胞在人体内, 以促进细胞到细胞的通信进程 1, 2.有趣的是, 外来体的组成会根据原始单元格的变化以及单个3、4、5的健康状况而改变.此外, 外来体可以从几种生物液中提取, 如: 唾液、尿液和血液 2.由于这些特点, 外来体被认为是疾病生物标志物的良好来源。不幸的是, 没有标准的方法来隔离外来体。一些实验室考虑多级离心, 以 10万 x g 的最终高速步骤为外切隔离的金标准方法。然而, 最近的研究表明, 离心诱导外来体与可溶性蛋白和其他外来体的聚集, 除了影响外切完整性, 这两个可能会阻碍下游应用程序6, 7.外切隔离的其他常用方法包括但不限于: 以商业聚乙二醇 (PEG) 为基础的试剂、离心超滤和尺寸排除色谱法 (SEC) 沉淀。使用聚乙二醇 (PEG) 聚合物的商业试剂通过使外来体沉淀并形成颗粒而丰富了它们。使用这种聚合物的限制是残留的 PEG 聚合物和大量的可溶性非 exosomal 蛋白在最终产品的污染。超滤利用纤维素膜进行离心纯化浓缩囊泡;外来体保留在过滤器之上, 而较小的杂质和其他蛋白质通过膜 7, 8.就像其他方法一样, 离心超滤由于保留高水平的非 exosomal 蛋白, 包括蛋白质复合物和聚合体, 外来体的净化能力有限。最后, SEC 净化使用多孔树脂分离分子的大小。SEC 已经显示出有希望的结果, 通过捕获大多数的污染物蛋白和保持 exosomal 完整性, 克服了其他方法所遇到的大多数问题, 因为隔离是基于重力或低压系统7, 9。然而, 在 SEC 期间, 大蛋白质集料和脂蛋白的联合分离影响了最终外切制剂的纯度.虽然一些方法已经测试了外切纯化从细胞培养上清液和等离子7, 或仅等离子9, 11, 没有关于直接比较血液的方法的性能信息同一个人的血浆和血清。
在这里, 我们重点研究血液外来体的纯化, 通过比较各种工作流, 以确定是否囊泡浓缩技术是可翻译的血浆和血清。采用纳米微粒跟踪分析和印迹方法, 对最终产物中外切浓度、纯度和蛋白质组成的差异进行量化。最后的方法, 详细的在本协议中, 增加囊泡数和减少非 exosomal 蛋白水平。重要的是, 大量减少常见的共沉淀蛋白, 包括白蛋白和载脂蛋白。这两种蛋白质在血液中的高丰度以及它们与外来体共同纯化的频率导致了 "纯化" 样品之间的不一致, 从而扭曲了下游的分析结果。该协议包括使用商业上可用的 SEC 树脂;该树脂是由多孔珠组成, 其中蛋白质小于 700 kDa 可以进入珠。一旦内, 蛋白质由辛胺配体保留。洗脱液由外来体和大于700kDa 12 的分子组成。此外, 为了减少载脂蛋白的蛋白质聚集和抑制, 我们在工作流中加入了蛋白酶消化步骤。目前, 还没有单一的技术能够最大限度地提高外切产量, 同时减少共纯化非 exosomal 蛋白。本研究表明, 采用蛋白酶消化预处理与多种回收纯化相结合的纯化方法, 可提高血清和血浆外切的产量和纯度。

<table><tbody><tr><td>Nanosight</td><td>Malvern</td><td>NS300</td><td></td></tr><tr><td>Benchtop microcentrifuge&amp;nbsp;</td><td>Thermo</td><td>Legend Mico21</td><td></td></tr><tr><td>Benchtop centrifuge&amp;nbsp;</td><td>Beckman Coulter</td><td>Allegra 6R</td><td></td></tr><tr><td>Waterbath&amp;nbsp;</td><td>Benchmark</td><td>B2000-4</td><td></td></tr><tr><td>Microplate reader</td><td>BIOTEK</td><td>Epoch</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>THERMO</td><td>23227</td><td></td></tr><tr><td>Micro BCA Protein Assay Kit</td><td>THERMO</td><td>23235</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>96 well plate</td><td>Corning</td><td>15705-066</td><td></td></tr><tr><td>Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-100 membrane</td><td>EMD-Millipore</td><td>UFC510096</td><td></td></tr><tr><td>Amicon Ultra-4 Centrifugal Filter Units</td><td>EMD-Millipore</td><td>UFC800324</td><td></td></tr><tr><td>Capto Core 700</td><td>GE Healthcare</td><td>17548103</td><td></td></tr><tr><td>Poly-Prep&amp;nbsp;Chromatography Columns</td><td>BIORAD</td><td>7311550</td><td></td></tr><tr><td>NuPAGE 4-12% Bis-Tris Protein Gels</td><td>THERMO</td><td>NP0323BOX</td><td></td></tr><tr><td>Nitrocellulose Membrane, Roll, 0.2 &amp;micro;m</td><td>BIORAD</td><td>1620112</td><td></td></tr><tr><td>Proteinase K,&amp;nbsp;&lt;em&gt;Tritirachium album&lt;/em&gt;</td><td>EMD-Millipore</td><td>539480</td><td></td></tr><tr><td>SimplyBlue SafeStain</td><td>THERMO</td><td>LC6065</td><td></td></tr><tr><td>SIGMAFAST&amp;nbsp;BCIP/NBT</td><td>Millipore-SIGMA</td><td>B5655</td><td></td></tr><tr><td>4-Chloro-1-naphthol</td><td>Millipore-SIGMA</td><td>C6788</td><td></td></tr><tr><td>Anti-CD63 Antibody (rabbit anti-human) with goat anti-rabbit HRP secondary antibody</td><td>SYSTEM BIOSCIENCES</td><td>EXOAB-CD63A-1</td><td>Primary and secondary antibodies</td></tr><tr><td>ALB Antibody (F-10)</td><td>SANTA CRUZ BIOTECHNOLOGY, INC.</td><td>sc-271605</td><td>Primary antibody</td></tr><tr><td>apoB Antibody (C1.4)</td><td>SANTA CRUZ BIOTECHNOLOGY, INC.</td><td>sc-13538</td><td>Primary antibody</td></tr><tr><td>apoA-I Antibody (B-10)</td><td>SANTA CRUZ BIOTECHNOLOGY, INC.</td><td>sc-376818</td><td>Primary antibody</td></tr></tbody></table>

protein digestion, ultrafiltration, and size exclusion chromatography to optimize the isolation of exosomes from human blood plasma and serum

外来体是一种从所有细胞类型中释放出来的 nanovesicle, 可以从任何体液中分离出来。外来体的内容, 包括蛋白质和 rna, 对它们所派生的细胞是独一无二的, 可以作为疾病的指标。一些常见的浓缩协议, 包括离心, 产外来体可溶性蛋白污染物。具体地说, 我们发现血液中最丰富的蛋白质经常与外来体共同纯化, 并可能混淆下游蛋白质组研究, 挫败低丰度生物标志物候选者的识别。另外的关注是由于非 exosomal 蛋白水平的不一致表示, 外切蛋白量化 irreproducibility。这里详述的协议是为了去除与外来体共纯化的非 exosomal 蛋白, 增加了外切纯化过程的严格。采用五例献血者配对血浆和血清对五种方法进行比较。用纳米微粒跟踪分析和微二辛可宁酸蛋白检测结果表明, 采用超滤和粒径排除色谱相结合的复合协议, 可获得最佳的囊泡浓缩和可溶性蛋白去除。西方印迹被用来验证预期丰富的血液蛋白, 包括白蛋白和载脂蛋白, 已耗尽。

以下数据是使用五健康献血者配对血浆和血清样本获得的。为了确定各处理步骤的效果, 对所提出的协议进行了五种变异, 并对外切恢复和非外切蛋白去除进行了比较。方法全身扫描仪最包括: 1) 秒 700 kDa + 超滤 3 kDa;2) 超滤 100 kDa;3) 蛋白酶 K + 超滤 100 kDa;4) SEC 700 kDa + 超滤 100 kDa, 5) 蛋白酶 K + 超滤 100 kDa + 秒 700 kDa + 超滤 3 kDa。
总...

Watch this Scientific Journal Video about Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum at JoVE.com

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

在这里, 我们提出了一个协议, 以净化外来体从血浆和血清与减少共同纯化的非 exosomal 血液蛋白。优化的协议包括超滤、蛋白酶处理和尺寸排除色谱。增强纯化外来体的好处下游分析, 包括更准确的定量的囊泡和蛋白质组特征。

蛋白质消化、超滤和尺寸排除色谱法优选人血浆和血清外来体的分离

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

protein-digestion-ultrafiltration-size-exclusion-chromatography-to

Research

JoVE Journal

Biochemistry

16.4K 次观看.  Colorado State University. 在这里, 我们提出了一个协议, 以净化外来体从血浆和血清与减少共同纯化的非 exosomal 血液蛋白。优化的协议包括超滤、蛋白酶处理和尺寸排除色谱。增强纯化外来体的好处下游分析, 包括更准确的定量的囊泡和蛋白质组特征。

视频: Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

从HIV-1阳性者的血浆分离外来体的

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

科研

免疫与感染

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the physiological and pathological state of a cell. However, there is no standard method for sEV isolation, which prevents downstream biomarker identification and drug intervention studies. In this article, we provide a detailed protocol for the isolation and purification of 50-200 nm sEV by a flow cell sorter. For this, a 50 &#181;m nozzle and 80 psi sheath fluid pressure were selected to obtain a good sorting rate and stable side stream. Standard sized polystyrene microspheres were used to locate populations of 100, 200, and 300 nm particles. With additional optimization of the voltage, gain, and forward scatter (FSC) triggering threshold, the sEV signal could be separated from the background noise. These optimizations provide a panel of critical sort settings that enables one to obtain a representative population of sEV using FSC vs. side scatter (SSC) only. The flow cytometry-based isolation method not only allows for high-throughput analysis but also allows for synchronous classification or proteome analysis of sEV based on the biomarker expression, opening numerous downstream research applications.

This protocol provides a rapid and size-specific isolation method for small extracellular vesicles by optimizing the size of the air spray nozzle, sheath fluid pressure, sample flow pressure, voltage, gain, and triggering threshold parameters.

小细胞外囊泡高通量分离纯化的流式细胞术分选参数优化

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the ...

生物化学

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

分离和剖析人力胆汁含外来体的MicroRNA-

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

医学

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

RNA含外来体的分离和鉴定

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

生物学

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

高产细胞外囊泡制剂的纯化远离病毒

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

对于外来体定量和尺寸测量新方法研究

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain proteins, nucleic acids, and lipids that are reflective of the parent cell from which they are derived. Proper separation of EVs from other components in a sample allows for characterization of their associated cargo and lends insight into their potential as intercellular communicators and non-invasive biomarkers for numerous diseases. In the current study, oligodendrocyte derived EVs were isolated from cell culture media using a combination of state-of-the-art techniques, including ultrafiltration and size exclusion chromatography (SEC) to separate EVs from other extracellular proteins and protein complexes. Using commercially available SEC columns, EVs were separated from extracellular proteins released from human oligodendroglioma cells under both control and endoplasmic reticulum (ER) stress conditions. The canonical EV markers CD9, CD63, and CD81 were observed in fractions 1-4, but not in fractions 5-8. GM130, a protein of the Golgi apparatus, and calnexin, an integral protein of the ER, were used as negative EV markers, and were not observed in any fraction. Further, when pooling and concentrating fractions 1-4 as the EV fraction, and fractions 5-8 as the protein fraction, expression of CD63, CD81, and CD9 in the EV fraction was observed. The expression of GM130 or calnexin was not observed in either of the fraction types. The pooled fractions from both control and ER stress conditions were visualized with transmission electron microscopy and vesicles were observed in the EV fractions, but not in the protein fractions. Particles in the EV and protein fractions from both conditions were also quantified with nanoparticle tracking analysis. Together, these data demonstrate that SEC is an effective method for separating EVs from conditioned cell culture media.

The protocol here demonstrates that extracellular vesicles can be adequately separated from conditioned cell culture media using size exclusion chromatography.

用于从条件细胞培养基中分离细胞外囊泡的体积排阻色谱

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain ...

神经科学

Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for diagnosis and prognosis or may serve as targets for drug development, respectively. Recently it has been shown that miRNAs are carried in lipoproteins, particularly high density lipoproteins (HDL) and are delivered to recipient cells for uptake. This raises the possibility that miRNAs play a critical and pivotal role in cellular and organ function via regulation of gene expression as well as messenger for cell-cell communications and crosstalk between organs. Current methods for miRNA isolation from purified HDL are impractical when utilizing small samples on a large scale. This is largely due to the time consuming and laborious methods used for lipoprotein isolation. We have developed a simplified approach to rapidly isolate purified HDL suitable for miRNA analysis from plasma samples. This method should facilitate investigations into the role of miRNAs in health and disease and in particular provide new insights into the variety of biological functions, outside of the reverse cholesterol transport, that have been ascribed to HDL. Also, the miRNA species which are present in HDL can provide valuable information of clinical biomarkers for diagnosis of various diseases.

MicroRNAs play an important regulatory role and are emerging as novel therapeutic targets for various human diseases. It has been shown that miRNAs are carried in high density lipoproteins. We have developed a simplified method to rapidly isolate purified HDL suitable for miRNA analysis from human plasma.

从高纯高密度脂蛋白的miRNA的高通量快速分离和简化方法

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for ...

Isolation and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can modulate the cellular status of other cells locally in tissue microenvironments or through liberation into peripheral blood. Adipocyte-derived EVs are elevated in the peripheral blood and show alterations in their cargo (RNA and protein) during metabolic disturbances, including obesity and diabetes. Adipocyte-derived EVs can regulate the cellular status of neighboring vascular cells, such as endothelial cells and adipose tissue resident macrophages to promote adipose tissue inflammation. Investigating alterations in adipocyte-derived EVs in vivo is complex because EVs derived from peripheral blood are highly heterogenous and contain EVs from other sources, namely platelets, endothelial cells, erythrocytes and muscle. Therefore, the culture of human adipocytes provides a model system for the study of adipocyte derived EVs. Here, we provide a detailed protocol for the extraction of total small EVs from cell culture media of human gluteal and abdominal adipocytes using filtration and ultracentrifugation. We further demonstrate the use of Nanoparticle Tracking Analysis (NTA) for quantification of EV size and concentration and show the presence of EV-protein tumor susceptibility gene 101 (TSG101) in the gluteal and abdominal adipocyte derived-EVs. Isolated EVs from this protocol can be used for downstream analysis, including transmission electron microscopy, proteomics, metabolomics, small RNA-sequencing,&#160;microarrays and can be&#160;utilized in functional in vitro/in vivo studies.

We describe the isolation of human adipocyte-derived extracellular vesicles (EVs) from gluteal and abdominal adipose tissue using filtration and ultracentrifugation. We characterize the isolated adipocyte-derived EVs by determining their size and concentration by Nanoparticle Tracking Analysis and by western blotting for the presence of EV-protein tumor susceptibility gene 101 (TSG101).

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can ...

<meta charset="utf8"></head><body>使用血浆细胞外囊泡蛋白质组的心血管生物标志物研究

Isolation, Characterization, and Proteomic Analysis of Plasma-Derived Extracellular Vesicles for Cardiovascular Biomarker Discovery

Extracellular vesicles (EV) are cell-derived, lipid bilayer-enclosed, non-replicable nanoparticles. EV currently gain attention in cardiovascular research due to their role in regulating intercellular communication, potentially serving as valuable biomarkers for cardiovascular disease. However, the EV proteome and its potential as a biomarker in cardiovascular diagnostics remain poorly understood. This protocol presents a standardized method for the isolation and quantification of plasma-derived EV and the analysis of their protein cargo using plasma samples from patients presenting to the Chest Pain Unit of a large university hospital. Following routine phlebotomy, EV are isolated from plasma by differential ultracentrifugation. The enrichment of specific EV marker proteins in EV isolates is visualized by immunoblotting, and average size distribution and plasma EV concentrations are quantified by nanoparticle tracking analysis. Finally, ultra-performance liquid chromatography-tandem mass spectrometry is employed for label-free analysis of the EV proteome. This protocol thus provides a comprehensive approach to study and use plasma-derived EV as potential carriers of critical biological information as well as to explore their potential as novel biomarkers.

<meta charset="utf8"></head><body>作者聚焦：推进血浆细胞外囊泡蛋白质组分析用于心血管生物标志物研究

This article describes a methodology for the isolation, characterization, and quantification of human plasma-derived extracellular vesicles (EV) and presents a workflow for label-free analysis of the EV proteome using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

血浆来源的细胞外囊泡的分离、表征和蛋白质组学分析用于心血管生物标志物的发现

Extracellular vesicles (EV) are cell-derived, lipid bilayer-enclosed, non-replicable nanoparticles. EV currently gain attention in cardiovascular research ...