Name: isolation and analysis of traceable and functionalized extracellular vesicles from the plasma and solid tissues
Uploaded: 2022-10-17T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues at JoVE.com

这项工作得到了中国国家自然科学基金（32000974，81870796，82170988和81930025）和中国博士后科学基金（2019M663986和BX20190380）的资助。我们感谢国家基础医学实验教学示范中心（AMFU）的帮助。

动物实验按照《第四军医大学机构动物护理使用委员会指南》和《ARRIVE指南》进行。在本研究中，使用8周龄的C57Bl / 6小鼠（对雌性或雄性都没有偏好）。分离血浆和组织EV所涉及的步骤如图 1所示。血浆以代表描述EV从体液中分离的过程。以上颌骨为代表，解释EV与实体组织的分离过程。
1. 血浆和上颌骨样本的制备
<ol><li>按照以下步骤制备?...

<ol>
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在研究电动汽车的特性、命运和功能时，隔离高产量和低污染的电动汽车至关重要。有多种方法可以提取 EV，例如密度梯度离心 （DGC）、体积排阻色谱 （SEC） 和免疫捕获测定4，20。这里使用了最常用的方法之一，差速离心;这样做的优点是不耗时，它可以产生高产量的EV，易于从有限的样品中分离出来，并且能够根据所使用的样品源修改方法。为了分?...

已经提出细胞外囊泡（EV）来定义细胞释放的脂质双层封闭的细胞外结构1，其在各种生理和病理事件中起重要作用2。健康细胞释放的EV大致可分为两大类，即通过细胞内内吞运输途径3形成的外泌体（或小EV）和细胞质膜向外出芽形成的微囊泡（或大EV）4。虽然许多研究集中在体外5中从培养细胞中收集的EV的功能，但来自循环或组织的EV更加复杂和异质，其优点是反映了体内生物体的真实状态6。此外，几乎所有种类的组织都可以在体内产生EV，这些EV可以作为组织内的信使或通过各种体液（尤其是外周血）转移，以促进全身交流7。循环和组织中的电动汽车也是疾病诊断和治疗的目标8。
近年来，外泌体的研究深入，微囊泡也具有重要的生物学功能，无需超速离心即可轻松提取，从而促进了基础和临床研究9。值得注意的是，关于从循环和组织中分离的EV的一个关键问题是它们来自不同的细胞类型10。由于微囊泡从质膜中吹出并具有丰富的细胞表面分子9，因此使用亲本细胞膜标记物来鉴定这些EV的细胞起源是可行的。具体而言，流式细胞术（FC）技术可用于检测膜标志物。此外，研究人员可以分离电动汽车并根据功能货物进行进一步分析。
本协议为从体内样品中提取和表征EV提供了全面的程序。EV通过差速离心分离，EV的表征包括通过纳米颗粒跟踪分析（NTA）和透射电子显微镜（TEM）进行形态学鉴定，通过FC进行原产地分析，以及通过蛋白质印迹进行蛋白质货物分析。以小鼠的血浆和上颌骨为代表。研究人员可以参考其他来源的电动汽车的该协议并进行相应的修改。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4% paraformaldehyde&#160;</td>
			<td>Biosharp</td>
			<td>143174</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>Alexa fluor 488 anti-goat secondary antibody</td>
			<td>Yeason</td>
			<td>34306ES60</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Alexa fluor 488 anti-rabbit secondary antibody</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Anti-CD18 antibody</td>
			<td>Abcam</td>
			<td>ab131044</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Anti-CD81 antibody</td>
			<td>Abcam</td>
			<td>ab109201</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>anti-CD9 antibody</td>
			<td>Huabio</td>
			<td>ET1601-9</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Anti-Mitofilin antibody</td>
			<td>Abcam</td>
			<td>ab110329</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>APOA1 Rabbit pAb</td>
			<td>Abclone</td>
			<td>A14211</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>BCA protein assay kit</td>
			<td>TIANGEN</td>
			<td>PA115</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>BLUeye Prestained Protein Ladder</td>
			<td>Sigma-Aldrich</td>
			<td>94964-500UL</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>MP Biomedical</td>
			<td>218072801</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Caveolin-1 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-53564</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>CellMask Orange plasma membrane stain</td>
			<td>Invitrogen</td>
			<td>C10045</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Chemiluminescence</td>
			<td>Amersham Biosciences</td>
			<td>N/A</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Curved operating scissor</td>
			<td>JZ Surgical Instrument</td>
			<td>J21040</td>
			<td>EV isolation</td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>Zhi Ke</td>
			<td>ZK-DST</td>
			<td>EV isolation</td>
		</tr>
		<tr>
			<td>Epoch spectrophotometer</td>
			<td>BioTek</td>
			<td>N/A</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Eppendorf tubes</td>
			<td>Eppendorf</td>
			<td>3810X</td>
			<td>EV isolation</td>
		</tr>
		<tr>
			<td>Flotillin-1 antibody</td>
			<td>PTM BIO</td>
			<td>PTM-5369</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Gel imaging system</td>
			<td>Tanon</td>
			<td>4600</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Golgin84</td>
			<td>Novus</td>
			<td>nbp1-83352</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Grids - Formvar/Carbon Coated - Copper 200 mesh</td>
			<td>Polysciences</td>
			<td>24915</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>Heparin Solution</td>
			<td>StemCell&#160;</td>
			<td>7980</td>
			<td>EV isolation</td>
		</tr>
		<tr>
			<td>Liberase Research Grade</td>
			<td>Sigma-Aldrich</td>
			<td>5401127001</td>
			<td>EV isolation</td>
		</tr>
		<tr>
			<td>Microscopic tweezer</td>
			<td>JZ Surgical Instrument</td>
			<td>JD1020</td>
			<td>EV isolation</td>
		</tr>
		<tr>
			<td>NovoCyte flow cytometer</td>
			<td>ACEA</td>
			<td>N/A</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Omni-PAGE Hepes-Tris Gels Hepes 4~20%, 10 wells</td>
			<td>Epizyme</td>
			<td>LK206</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>OSCAR(D-19) antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-34235</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>PBS (2x)</td>
			<td>ZHHC</td>
			<td>PW013</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Sigma-Aldrich</td>
			<td>57-33-0</td>
			<td>Anesthetization</td>
		</tr>
		<tr>
			<td>Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L)</td>
			<td>Jacson</td>
			<td>115-035-003</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L)</td>
			<td>Jacson</td>
			<td>111-035-003</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Phosphotungstic acid</td>
			<td>RHAWN</td>
			<td>12501-23-4</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>PKM2(d78a4) xp rabbit&#160; mab&#160;</td>
			<td>Cell Signaling</td>
			<td>4053t</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Polyethylene (PE) film</td>
			<td>Xiang yi</td>
			<td>200150055</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>Polyvinylidene fluoride membranes&#160;</td>
			<td>Roche</td>
			<td>3010040001</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Protease inhibitors</td>
			<td>Roche</td>
			<td>4693132001</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Recombinant anti-PGD antibody</td>
			<td>Abcam</td>
			<td>ab129199</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>RIPA lysis buffer</td>
			<td>Beyotime</td>
			<td>P0013</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>SDS-PAGE loading buffer (5x)</td>
			<td>Cwbio</td>
			<td>CW0027S</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Size beads</td>
			<td>Invitrogen</td>
			<td>F13839</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Tabletop High-Speed Micro Centrifuges</td>
			<td>Hitachi</td>
			<td>CT15E</td>
			<td>EV isolation</td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>HITACHI</td>
			<td>H-7650</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>MP Biomedicals</td>
			<td>19472</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Vortex Mixer Genie</td>
			<td>Scientific Industries</td>
			<td>SI0425</td>
			<td>EV isolation</td>
		</tr>
		<tr>
			<td>ZetaView BASIC NTA - Nanoparticle Tracking Video Microscope PMX-120</td>
			<td>Particle Metrix</td>
			<td>N/A</td>
			<td>Nanoparticle tracking analysis</td>
		</tr>
		<tr>
			<td>&#945;-Actinin-4 Rabbit mAb</td>
			<td>Abclone</td>
			<td>A3379</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>&#946;-actin</td>
			<td>Cwbio</td>
			<td>CW0096M</td>
			<td>Western blot</td>
		</tr>
	</tbody>
</table>

isolation and analysis of traceable and functionalized extracellular vesicles from the plasma and solid tissues

循环和组织驻留的细胞外囊泡（EVs）作为新型治疗诊断生物标志物代表了有希望的靶标，它们成为维持机体稳态和广泛疾病进展的重要参与者。虽然目前的研究重点是具有内体起源的内源性外泌体的特征，但从质膜中起泡的微囊泡在健康和疾病中越来越受到关注，其特征是丰富的表面分子概括了亲本细胞的膜特征。在这里，提出了一种基于差速离心的可重复程序，用于从血浆和实体组织（例如骨骼）中提取和表征EV。该协议进一步描述了电动汽车表面抗原和蛋白质货物的后续分析，因此可以追溯其衍生物，并识别与潜在功能相关的组件。该方法将有助于在生物学、生理学和病理学研究中对 EV 进行相关、功能和机理分析。

根据实验工作流程，可以从外周血和实体组织中提取EV（图1）。8周龄小鼠的上颌骨约为0.1±0.05g，可以从小鼠中收集约300μL血浆。按照协议步骤，可以分别收集0.3 mg和3 μg的EV。通过TEM和NTA分析，EV的典型形态特征是直径范围为50-300nm的圆形杯形膜囊泡（图2）。FC可以在有用的对照下检测膜染料标记的EV，FC分析显示了EV上表达的特定膜标志物的百分比，这...

Watch this Scientific Journal Video about Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues at JoVE.com

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

本协议描述了一种从外周血和实体组织中提取细胞外囊泡的方法，随后对表面抗原和蛋白质货物进行分析。

从血浆和实体组织中分离和分析可追溯和功能化的细胞外囊泡

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important ...

该协议提供了一种可行的方法来从大多数血液和实体组织中分离和收集正确的细胞外囊泡，特别是分析其来源和蛋白质含量，这有助于进一步的功能实验。除了高产量和低污染外，该协议的主要优点是分析EV的表面抗原和蛋白质货物，这对于生理和病理学研究很有用。它通过使用流式细胞仪检测细胞外囊泡亲本细胞的表面标志物，促进疾病癌症的诊断，例如炎症性疾病和骨质疏松症。组织细胞外囊泡的数量对于以下实验至关重要。确保细胞外囊泡的浓度不高。耐心等待稀释。首先，通过用眼科镊子和剪刀分离上颌骨来制备上颌骨样本，并用PBS清洗它们以用镊子去除软组织。然后将上颌骨放入1.5毫升离心管中，用剪刀将骨头切成直径一毫米的小块。加入一定量的解放酶覆盖组织，在37摄氏度下孵育30分钟。接下来，在4摄氏度下以800 G离心样品10分钟，并用移液管小心地将上清液转移到新的清洁的1.5毫升离心管中。将制备的血浆和骨样品在2， 500 G和4摄氏度下离心15分钟以除去大细胞碎片和剩余的血小板后，小心地将上清液转移到新的和干净的1.5毫升离心管中，并在4摄氏度下以16， 800 G离心30分钟。接下来，弃去上清液，并用一毫升PBS重悬在每个管中。如前所述离心并丢弃上清液后，再次用50微升PBS将沉淀重悬于每个管中，并将这些样品在4摄氏度下储存少于24小时或最好立即使用它们进行分析。用500微升PBS重悬样品，并将50微升转移到新的干净的1.5毫升离心管中，作为标记为管A的空白对照，将另外50微升转移到另一个干净的1.5毫升离心管中作为FITC的简单染色管，标记为管B.向原管中加入0.5微升膜染料进行膜染色，同时在室温下孵育五分钟。将原管以16，800G离心30分钟并弃去上清液后，用200微升PBS重悬沉淀。将每个 50 μL 样品分成四个 1.5 毫升离心管，用于标记为管 C 的 PE 的简单染色管、标记为管 D 的表面标志物染色和标记为试管 E 的仅二抗对照.将破骨细胞相关受体和骨细胞外囊泡样品的一抗以及 CD-18 抗体和血浆细胞外囊泡样品分别添加到试管 B 和试管 D 中， 在四摄氏度下孵育一小时。如前所述离心管并丢弃上清液，并用500微升PBS重悬沉淀。同样，在16，800 G和4摄氏度下离心30分钟以除去额外的一抗。丢弃上清液后，用50微升PBS重悬沉淀，然后分别在管E中加入FITC偶联的二抗.将所有管在黑暗中以4摄氏度孵育一小时。将一滴0.2、0.5和1微米大小的珠悬浮液分别稀释到一毫升PBS中。然后运行每种尺寸的磁珠以确保为细胞外囊泡选择的门，并设置流式细胞仪的阈值，以使用合适的前向内散射搜索磁珠和细胞外囊泡群。将终端条件设置为计算 100， 000 个膜染色颗粒，并按照手稿中所述通过流式细胞仪分析样品。将沉淀重悬于50微升RIPA裂解缓冲液中，并在冰上孵育30分钟。为了通过BCA蛋白质测定定量96孔微孔板中所有样品的蛋白质浓度，将每毫升2毫克BSA与0.9%的生理盐水稀释成0.5毫克/毫升。以每毫升0.5毫克BSA和0.9%的生理盐水进入三个具有一定体积的重复孔中。然后滴下两微升样品，并将18微升生理盐水分别加入三个重复的孔中。通过将BCA试剂A与试剂B混合制备工作溶液后，向每个孔中加入200微升工作溶液并轻轻摇动30秒。然后将96孔微孔板在37摄氏度下孵育20至25分钟，并用分光光度计测量596纳米处的光密度。接下来，导出数据，绘制标准曲线，并计算样品的蛋白质浓度。根据结果，用0.9%的生理盐水和五倍的SDS-PAGE上样缓冲液将样品稀释至每微升1微克，并用薄膜密封试管并在100摄氏度下加热五分钟。将蛋白质分子量标准中的样品加载到4至20%HEPy的tris凝胶的梯度浓度中。在电泳缓冲液中以80伏电泳凝胶，直到蛋白质形成一条线。然后切换到 120 伏 1 小时，直到上样染料位于凝胶底部。使用湿转印系统将凝胶转移到在甲醇中预孵育20秒的聚偏氟乙烯膜上，以200毫安的电流转移一小时。通过将 2.5 克 BSA 和 50 毫升 TBST 加入 50 毫升离心管中来制备 5% BSA 封闭缓冲液。在室温下搅拌将该缓冲液中的膜封闭两小时。用TBST稀释的特异性一抗孵育膜，使其在4摄氏度下达到适当的浓度过夜。在洗涤缓冲液中洗涤膜四次，并在室温下搅拌将膜与合适的二抗孵育一小时。在洗涤缓冲液中洗涤膜四次后，在凝胶成像系统中使用化学发光试剂盒对膜进行成像。透射电镜和纳米颗粒跟踪分析表明，细胞外囊泡的典型形态特征为圆形和杯形，直径范围为500-300纳米。流式细胞术分析显示细胞外囊泡上表达的特定膜标志物的百分比，这意味着它们来自亲本细胞。蛋白质印迹分析显示PGD和PKM2的表达分别代表蛋白质含量和血浆细胞外囊泡和骨细胞外囊泡，表明代谢状态。在细胞外囊泡中检测到Golgin-84的阴性表达，存在丝裂蛋白，α-肌动蛋白-4，氟氏蛋白-1，Caveolin-1和β-肌动蛋白以及囊泡标志物CD9，而CD81表达低或不存在，血浆细胞外囊泡中缺乏APO-A-1。研究人员，所以请记住，步骤2.2，组织样品的制备和消化应充分完成。否则，从组织中提取的细胞外囊泡的数量是有限的。标记的细胞外囊泡荧光染色剂可以注射到口腔中以治疗与潜在功能相关的阶段。

Research

JoVE Journal

Biology

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

从胎盘中的造血干细胞的分离和分析

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

科研

生物学

Solid Phase Synthesis of a Functionalized Bis-Peptide Using "Safety Catch" Methodology

一个官能二肽的固相合成使用“安全锁扣”方法论

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

对于外小泡的使用流式细胞仪分析技术

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

从富集组织及消化细胞外基质蛋白的成肽质谱分析

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

细胞调理介质细胞外囊泡的流式细胞学分析

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

卡诺尔哈布炎细胞外囊泡的纯化与分析

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

细胞外囊泡的量化和尺寸测定纳米粒子跟踪分析

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving ...

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...