Name: flow cytometric analysis of extracellular vesicles from cell-conditioned media
Uploaded: 2019-02-12T14:00:00
Description: Watch this Scientific Journal Video about Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media at JoVE.com

l. b. 得到了苏黎世 (瑞士) helmut horten stiftung 和 velux stiftung 的研究赠款的支持。g. v. 得到瑞士国家科学基金会、cecilia-奥古斯塔基金会、lugano 和 shk stiftung für herz-und kreislaufkrankheiten (瑞士) 的研究赠款的支持。

1. 有条件介质的收集和处理
<ol><li>在 pbs中涂上55厘米2培养皿, 含有0.02% 的猪皮明胶。</li>
	<li>板 cpc (8, 000/cm 2) 在预涂层的菜肴与7毫升的 iscove 的改性 dulbecco 的培养基 (imdm) 补充 20% fbs (胎儿牛血清) 和 1% penicililin/strepmicin (p/s). 请注意:"cpc" 一词是指其他地方所描述的人类外植体衍生细胞。cm 可以从在特定培养条件下培养的不同细胞类型中收集。戴上...

<ol>
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传统的 fc 技术仍然是表征 ev 表面标记的最直接的分析方法。在这方面, 选择最适当的协议对于通过避免因仪器灵敏度而造成的限制, 获取有关感兴趣的单个粒子分数的有用信息至关重要。我们描述了一种方法, 使用磁性颗粒结合抗体, 识别 exo 和小 ev 表面抗原, 适合下游 fc 应用。我们使用两种不同的细胞类型验证了这种方法: 初级人类 cpc 正在成为基于 exo 的心脏病治疗方法的主要细胞来源;hek293 细?...

细胞分泌不同大小的细胞外囊泡 (ev), 包括微囊泡 (mv) 和外显子体 (exo)。后者可以通过大小和源亚细胞隔间来区别于 mv。mv (200–1000 nm 大小) 通过从质膜脱落从母细胞中释放。相反, exo (30–150 nm) 起源于子宫内膜, 当多泡体 (mvb) 与细胞膜1,2融合时, exo (30–150 nm) 会释放到细胞外空间。
ev 越来越多地被用作诊断生物标志物, 并可能被用作许多领域的治疗工具, 包括肿瘤学、神经病学、心脏病和肌肉骨骼疾病3, 4,5。绝大多数正在进行的使用 ev 作为治疗药物的研究利用从细胞条件下的细胞培养基 (cm) 的离体外培养细胞 (cm) 中分离出的小泡。间充质干细胞 (mscs) 在多种情况下发挥着有益的作用, msc 衍生的 ev 在心肌缺血再灌注损伤6和脑损伤7的模型中表现出了有益的效果。msc 衍生的 ev 还表现出免疫调节活性, 可用于治疗免疫排斥, 如治疗难治性移植物抗宿主病模型8所示。羊水干细胞 (hafs) 用 mv 和 exo 积极丰富 cm, 在大小 (50–1000 nm) 上分布不均匀, 介导了几种生物效应, 如分化细胞增殖、血管生成、纤维化抑制、心脏保护4。我们最近已经证明, ev, 特别是 exo, 由人类心源性祖细胞 (exo-cpc) 分泌减少心肌梗死大小的大鼠 5,9。
exo 在其表面共享一组常见的蛋白质, 包括四帕辛 (cd63、cd81、cd9) 和主要组织相容性复合体 i 类 (mhc-i)。除了这一组常见的蛋白质外, exo 还包含特定于生产者细胞类型的 ev 子集的蛋白质。exo 标记越来越重要, 因为它们在细胞间通信中起着至关重要的作用, 从而调节了许多生物过程5,10。由于电动汽车体积小, 找到一种使用经典流式细胞仪 (fc) 分析 ev 的简单方法仍然是一项具有挑战性的任务。
在这里, 我们提出了一个使用 fc 进行 ev 分析的简化协议, 该协议可应用于通过超离心或直接应用于 cm 获得的预浓缩样品 (图 1)。该方法使用带有特定抗体的珠子, 该抗体结合了规范的外源表面表位 (cd63、cd9、cd81), 无需额外清洗。fc 分析可以使用传统的细胞仪进行, 无需在测量前进行调整。其他群体在各种应用中描述了利用流式细胞仪对单个小颗粒进行抗原表征的方法11、12、13。在这里, 我们使用功能化磁珠捕获小颗粒和 exo, 然后由 fc 对捕获的粒子进行表型。虽然这种方法可以用来表征任何细胞在体外释放的小囊泡的抗原成分, 但在这里我们提供了适用于人类心脏祖细胞培养 (cpc) 和最多的特定细胞培养条件。适当的环境, 为这些细胞生产的 ev。

<table border="0" cellpadding="0" cellspacing="0" style="width:816px;" width="816">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:369px;">IMDM</td>
			<td style="width:113px;">Gibco</td>
			<td style="width:235px;">12440061</td>
			<td style="width:99px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra-15, PLHK Ultracel-PL Membran, 100&#160;kDa&#160;</td>
			<td>Millipore</td>
			<td>UFC910024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CytoFlex, Flow Cytometer Platform</td>
			<td>Beckman Coulter</td>
			<td>CytoFlex</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, high glucose, HEPES, no phenol red</td>
			<td>Gibco</td>
			<td>21063045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s PBS (PBS) Ca- and Mg-free</td>
			<td>Lonza</td>
			<td>BE17-512F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ExoCap CD63 Capture Kit</td>
			<td>JSR Life Sciences</td>
			<td>Ex-C63-SP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ExoCap CD81 Capture Kit</td>
			<td>JSR Life Sciences</td>
			<td>Ex-C81-SP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ExoCap CD9 Capture Kit</td>
			<td>JSR Life Sciences</td>
			<td>Ex-C9-SP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exosome-Depleted FBS</td>
			<td>Thermofisher</td>
			<td>A2720801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exosome-depleted FBS Media Supplement</td>
			<td>SBI</td>
			<td>EXO-FBS-250A-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS-Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC anti-human CD9 Antibody</td>
			<td>Biolegend</td>
			<td>312104&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; RRID: AB_2075894</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow Cytometer analysis software</td>
			<td>Beckman Coulter</td>
			<td>Kaluza</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoSight LM10</td>
			<td>Malvern</td>
			<td>NanoSight LM10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoSight Software</td>
			<td>Malvern</td>
			<td>NTA 2.3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optima Max-XP</td>
			<td>Beckman Coulter</td>
			<td>393315</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE anti-human CD63 Antibody</td>
			<td>Biolegend</td>
			<td>353004&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; RRID:AB_10897809</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE anti-human CD81 (TAPA-1) Antibody</td>
			<td>Biolegend</td>
			<td>349505&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; RRID:AB_10642024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermomixer C</td>
			<td>Eppendorf</td>
			<td>5382 000 015</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">TLA-110</td>
			<td>Beckman Coulter</td>
			<td>TLA-110 rotors</td>
			<td></td>
		</tr>
	</tbody>
</table>

flow cytometric analysis of extracellular vesicles from cell-conditioned media

流式细胞术 (fc) 是细胞表面抗原标志物半定量测量的首选方法。近年来, 该技术已被用于外周血和其他体液中的外细胞囊泡 (ev) (ev) 的表型分析。ev 体积小, 要求使用检测阈值在50-100 纳米左右的专用仪器。或者, ev 可以绑定到可由 fc 检测到的乳胶微珠。微珠, 与抗体结合, 识别与 ev 相关的标记--分化 cd63、cd9 和 cd81 的聚类可用于电动汽车捕获。从 cm 分离出的 exo 可以用超离心法进行预浓缩或不浓缩的分析。该方法适用于使用传统 fc 仪器的电动汽车分析。我们的结果表明, 平均荧光强度 (mfi) 值与 ev 浓度之间存在线性关系。通过超声破坏 ev 的 mfi 大幅减少, 表明该方法不能检测到膜碎片。我们报告了一种准确可靠的 ev 表面抗原分析方法, 可在任何实验室轻松实施。

单次染色的颗粒总数
由于单个珠子可以结合多个粒子, 我们测试了不同的条件, 以设置最小的总 ev (每个管的单抗体), 以达到 mfi 曲线的早期指数相。使用了固定浓度的抗体, 而粒子总数从 5 x 10 5 到 2.5 x10 8不等.如图 3a所示, 允许我们确保抗体在可接受的 mfi 中运行, 避免使用过?...

Watch this Scientific Journal Video about Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media at JoVE.com

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

该协议描述了一种可重复的方法, 该方法设计用于细胞培养上清液, 以检测小细胞外囊泡 (ev) 上的表面表位。它利用特定的 ev 免疫沉淀使用珠子与抗体结合, 识别表面抗原 cd9, cd63 和 cd81。该方法对下游流式细胞仪分析进行了优化。

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 ...

细胞外囊泡越来越多地被用作许多领域的诊断生物标志物和潜在的治疗工具。流细胞测量是描述适合面标记的最直接分析方法，本协议可用于分析一个超大颗粒，速度快，适用于传统的地震仪，无需特殊设置或专用仪器。虽然此协议用于从该条件和介质中对细胞外囊泡进行分类，但它可以扩展到血液到达的AV分类，以取代患者的侵入性组织活检。对于新进入该协议的研究人员来说，执行设置方法部分中描述的步骤，并密切遵循演示的获取策略至关重要。15分钟后，将盘子和盘子4.4倍10至第五个心脏祖细胞洗到每盘7毫升的伊斯科夫的改性Dulbecco的中、IMDM中，辅以20%的胎儿牛血清和1%青霉素和链霉素，用于在37摄氏度和5%二氧化碳的孵育。当细胞达到约80%的汇合，用Dulbecco的PBS洗两次，不含钙和镁，用10个无血清外体生产介质喂养细胞。7天后，将调节介质从每个板中放入一个聚丙烯离心管中，并离心介质以清除任何细胞碎屑。将超滤器转移到100千吨离心过滤管中，并通过额外的离心浓缩清除的、有条件的介质。将浓缩物转移到微型离心管中，进行最后的台式离心，并将超薄板加入聚碳酸酯厚壁微离心管。用PBS填充管，最终体积为3.2米，将样品装入钛固定天使转子中。然后将转子装入桌面超离心，进行超离心，并在 100 微升新鲜 PBS 中重新装入颗粒。对于纳米粒子跟踪和分析定量，在 999 微升 PBS 中稀释 1 微升的超离心样品，然后将样品加载到无气泡的 1 个米莱特注射器中。将注射器装入检查室的入口口，然后打开激光。使用捕获按钮打开相机，并调整对焦。然后记录至少三个不同的帧，每个帧 60 秒，并使用软件中的批处理选项，分析三个不同的采集。要准备样品进行采集，首先将适当的实验单克隆抗体结合捕获珠以1：1：1的比例加入微离心管中，然后涡旋产生的捕获珠池。接下来，将适当的样品体积添加到捕获珠池的 1 微升中，以生成 1 倍 10 到 8 颗粒加上 1.2 倍 10 到每个测试浓度的第 5 个珠子。将1微升珠子加入标有磁珠的管子作为负控制，并将每个管中的体积调整为100微升，并配有新鲜的PBS。然后将管子放入热混合器中，以每分钟 400 次旋转和 4 到 10 摄氏度的速度进行摇动。第二天，将样品转移到圆形底部96井板，并在每个井中加入适当的荧光结合抗体。在4至10摄氏度下孵育1小时后，每井加入100微升PBS，并打开采集软件。选择细胞仪和系统启动程序，启动仪器并打开新的实验。创建实验模板，并选择板并添加板。选择样品在板上的位置，然后单击设置为样本。在命名规则中输入示例名称，打开通道选项卡，然后选择适当的荧光信号通道。现在将板装入仪器。然后单击点图，以创建新的向前散点区域与侧散点区域点图。选择初始化以启动激光和流体，然后单击运行以开始采集。调整刻度以显示前进和侧散射图中间的群体，并围绕整个样本总体绘制一个珠门以排除碎片。单击"停止"和"点"图以创建向前散点高度与向前散点区域点图，并在珠子总体上对样本进行门控。绘制一个新的门围绕更大的人口和命名它单，并点击点图创建一个荧光信号与侧散点区域点图，每个实验荧光团使用。门点上的单点图。然后选择要记录的事件数，然后选择记录以开始实验采集。在分析结束时，将采集文件加载到相应的分析软件中，并打开新的协议。为每个文件创建点图（如刚刚演示的），并设置所有散点轴为线性比例，将所有荧光轴设置为日志比例。然后在相关信号通道中显示荧光几何均值，计算不同细胞外囊泡制剂中平均花度强度的全变化。在测试不同的条件，以设置最小的细胞外囊泡总量，以达到平均荧光强度曲线的早期指数相后，确定最小颗粒数为1倍10至8个颗粒每染色。抗体的最佳浓度为1倍10至8个颗粒，达到无非特异性抗体结合的最高信号，被确定为每毫升10微克，用于抗CD9_FITC和抗CD63_PE抗体，每毫升5微克用于抗CD81_PE抗体。为了确认该方法的适用性，为了分析杯形的细胞外囊泡，而不是膜碎片，对细胞外囊泡进行声波化，在经过30秒的声波后，平均荧光强度降低，在机械中断一分钟后，所有检查都未检测到荧光。X本身从心脏祖细胞的纯化，如证明，产生细胞外囊泡，表达低水平的CD9和高水平的CD63和CD81从两个细胞群体。此外，虽然 X 自己的表面标记在没有超离心的情况下可以清晰识别，但此浓缩步骤可大大增强这些标记的均度度强度。当处理细胞外囊泡时，很容易错过更粗心的纳米颗粒。因此，必须遵循协议中演示的所有步骤。使用细胞外囊泡作为生物标志物是目前最新兴的领域研究之一。很快，可以转化为临床诊断用作替代筛查工具。

Research

JoVE Journal

Biology

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

使用CD71/TER119流流式细胞仪检测小鼠红系祖细胞的识别和分析

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

科研

生物学

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 ...

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 ...

Isolation and Characterization of Cyanobacterial Extracellular Vesicles

蓝藻细胞外囊泡的分离和表征

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential ...

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many ...

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

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 ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

培养的人间充质干细胞细胞外囊泡的分离、表征和治疗应用

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...