Name: optimization of flow cytometric sorting parameters for high-throughput isolation and purification of small extracellular vesicles
Uploaded: 2023-01-20T14:50:00
Description: Watch this Scientific Journal Video about Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles at JoVE.com

本工作得到了浙江中医药大学科研基金（2020ZG29）、浙江省基础公益研究项目（LGF19H150006，LTGY23B070001）、浙江省教育厅项目（Y202147028）和浙江大学实验科技项目（SJS201712，SYB202130）的支持。

1. 细胞培养
<ol><li>准备补充有10%胎牛血清（FBS）的Dulbecco改良鹰培养基（DMEM）培养基。在75cm2 培养瓶中以1 x 106 个细胞/ mL的密度培养人胰腺癌细胞PANC-1。将培养物在37°C与5%CO2孵育。</li>
	<li>当细胞密度在显微镜观察下达到75%-80%时，传代培养细胞。取出培养基，用 3-5 mL 磷酸盐缓冲盐水 （PBS; 0.01 M， pH 7.2-7.4） 冲洗 2 次。</li>
	<li>弃去溶液，加入1-2mL胰蛋白酶-E...

<ol>
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该协议概述了使用流通池分选仪分离和纯化指定粒径为50-200 nm的sEV的优化方法，该方法已通过NTA验证。该方法解决了获得粒径均匀、纯度高的sEV的瓶颈问题，避免了包裹在大型EV中的无关生物分子的干扰22。流式细胞术可以进行快速、高通量分析，每秒可捕获 100，000 个颗粒，每秒做出 70，000 次分选决策17，大大减少了时间并反映了 sEV 颗粒的异质性。此外，这?...

细胞释放不同大小的细胞外囊泡（EV），产生信号分子和膜包涵体，这对细胞间通讯很重要1。不同大小的EV也起着不同的生物学作用，50-200nm sEV能够将RNA，DNA和蛋白质精确地分配到正确的细胞外位置。sEV还有助于确定其分泌机制，不仅涉及正常生理过程的调节，如免疫监视，干细胞维持，血液凝固和组织修复，还涉及肿瘤进展和转移等几种疾病的潜在病理学2，3。有效分离和分析sEV对于识别生物标志物和设计未来的药物干预措施至关重要。
随着sEV临床应用的不断研究，sEV的分离方法提出了更高的要求。由于sEV在大小、来源和含量上的异质性，以及它们在理化和生化性质上与其他EV的相似性，因此没有标准的方法进行sEV分离4，5。目前，超速离心、体积排阻色谱（SEC）、聚合物沉淀和免疫亲和捕获是最常见的sEV分离方法6。超速离心仍然是研究中sEV分离的金标准，尽管耗时，导致纯度低，尺寸分布为40-500 nm，并且在长期离心后对sEV造成重大机械损坏7，8，9。通常使用聚乙二醇（PEG）的聚合物沉淀在随后的功能分析中具有不可接受的纯度，同时细胞外蛋白质聚集体的沉淀和聚合物污染10，11。基于免疫亲和捕获的方法需要具有不同特异性的高成本抗体，并且存在处理量和产量低的问题12，13，14。粒径是评价分离sEV纯度的主要指标之一。尽管sEV纯度仍然是一个无法实现的目标，但SEC去除了相当数量的介质成分，并且通过SEC方法提取的sEV颗粒主要在50-200nm15的范围内。现有技术存在一些缺点，包括但不限于耗时、纯度低、产量低、重现性差、样品通量低以及sEV的潜在损害，这使得其与临床利用不兼容16。因此，适用于多种生物流体的快速、廉价且尺寸指定的sEV分离方法是多种研究和临床情况下的基本需求。
在流式细胞术中，以高通量、多参数的方式分析单个颗粒，并分选出亚群17.由于 EV 的异质性，单颗粒流式细胞术测量将是理想的，它已被用于按照细胞分析范式研究 EV，光散射和荧光标记用于识别生理学相关特征和蛋白质成分18，19，20。然而，传统的流式细胞术受到sEV体积小和表面生物标志物丰度低的挑战。无论使用前向散射（FSC）、侧向散射（SSC）还是荧光阈值触发参数19，都可以通过优化检测参数来区分背景噪声和sEV，从而提高流式细胞术的灵敏度。
使用本方案，使用荧光珠作为标准优化高分辨率流式细胞术分选设置。通过选择合适的喷嘴尺寸、护套流体压力、阈值触发参数和控制散射光强度的电压，我们能够从复杂的混合物中分离出 sEV 的特定子集。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Centrifuge tube</td>
			<td>Beckman Coulter</td>
			<td>344058</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture flasks</td>
			<td>Corning&#160;</td>
			<td>430641</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified eagle medium</td>
			<td>Corning Cellgro</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>SUER</td>
			<td>SUER050QY</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cell sorter</td>
			<td>Beckman Coulter</td>
			<td>Moflo Astrios EQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Human pancreatic cancer cell, PANC-1</td>
			<td>NA</td>
			<td>NA</td>
			<td>PANC-1 cells were donated by Professor Weijun Yang, College of Life Sciences, Zhejiang University</td>
		</tr>
		<tr>
			<td>Laser particle size and zeta potential analyzer&#160;</td>
			<td>Malvern</td>
			<td>Zetasizer Nano ZS 90</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer saline</td>
			<td>Gibco</td>
			<td>C20012500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene fluorescent microspheres</td>
			<td>Beckman Coulter</td>
			<td>6602336</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission electron microscopy</td>
			<td>JEOL</td>
			<td>JEM-1200EX</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Gibco</td>
			<td>1713949</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra rainbow fluorescent particles</td>
			<td>Beckman Coulter</td>
			<td>B28479</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Optima-L80XP</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge rotor</td>
			<td>Beckman Coulter</td>
			<td>SW32TI</td>
			<td></td>
		</tr>
	</tbody>
</table>

optimization of flow cytometric sorting parameters for high-throughput isolation and purification of small extracellular vesicles

小细胞外囊泡（sEV）可以从所有细胞类型中释放出来，并携带蛋白质，DNA和RNA。信号分子是细胞生理和病理状态的指标。然而，sEV分离没有标准方法，这阻碍了下游生物标志物鉴定和药物干预研究。在本文中，我们提供了通过流通池分选仪分离和纯化50-200 nm sEV的详细方案。为此，选择了50 μm喷嘴和80 psi鞘液压力，以获得良好的分选率和稳定的侧流。标准尺寸的聚苯乙烯微球用于定位100、200和300 nm颗粒的群体。通过进一步优化电压、增益和前向散射（FSC）触发阈值，可以将sEV信号与背景噪声分离。这些优化提供了一个关键排序设置面板，使人们能够仅使用 FSC 与侧向散射 （SSC） 获得具有代表性的 sEV 群体。基于流式细胞术的分离方法不仅可以进行高通量分析，还可以基于生物标志物表达对sEV进行同步分类或蛋白质组分析，从而开辟了许多下游研究应用。

实验方案的流程图如图 1所示。该方法以标准尺寸聚苯乙烯微球作为粒径分布的参考标准品。在特定仪器参数条件下，使用对数形式可以清楚地将粒子信号与FSC与SSC图中的背景噪声区分开来。门控策略如图 2 所示。R4、R5 和 R6 分别指 100 nm、200 nm 和 300 nm 微球的位置。R7是50nm以下电子噪声的检测限，R8是50-200nm粒子的位置范围。
超速?...

Watch this Scientific Journal Video about Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles at JoVE.com

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

该协议通过优化空气喷嘴的尺寸、鞘液压力、样品流压、电压、增益和触发阈值参数，为小细胞外囊泡提供了一种快速且尺寸特异性的分离方法。

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 ...

该协议通过优化流式细胞术分选参数，为小细胞外囊泡提供了一种快速且尺寸特异性的解决方案方法。这些优化提供了一个关键分类设置面板，使人们能够使用前向散射而不是仅使用大小散射获得具有代表性的小细胞外囊泡群体。首先执行流通池分选仪的标准启动程序。按启动按钮打开机器，然后单击激光控制面板上的激光电源按钮。按下智能进样器子菜单上的更换罐按钮，对流体进行加压。使用超声波清洁的 50 微米喷嘴和空气喷嘴，并将其安装到喷嘴组件上。在压力控制台上将鞘液的压力调节为 80 SI，将样品流量的压力调节为 80.3 SI。按下启动护套流按钮，启动护套流。点按“智能进样器”子菜单中的气泡按钮以自动去气泡 10 分钟，然后将其关闭。要对齐流并确定激光光斑，请打开照明室灯以查看针孔上的流。调整上下、左右、前后千分尺，使流在针孔上居中并聚焦流。打开所有激光快门，选择激光和流截距选项卡，然后根据提示连续按下绿色箭头按钮，完成激光截距和喷嘴对准校准过程。访问精细的激光对准屏幕。选择 640 纳米激光器和 772 bar 44 带通通道作为 x 轴参数，选择 405 纳米激光器和 448 bar 59 带通通道作为 y 轴参数。按下触发参数选择器，然后选择488纳米激光器的前向散射参数。接下来，通过将一滴加入一毫升双蒸水中至终浓度为每毫升 1 倍 10 至第六颗珠子来稀释彩虹荧光颗粒。打开样品室门并加载一管准备好的彩虹荧光颗粒。按下起始样品按钮并上下调整千分尺以优化荧光强度，使每个信号尽可能强烈。按下触摸屏控制面板上的开始质量控制或质量控制按钮以自动执行质量控制。然后按智能排序初始化按钮。仪器自动调整液滴振荡的频率和幅度，以获得大约25的液滴延迟，并能够清楚地看到液滴的断裂点。接下来，按下降延迟按钮和维护按钮。选择管子作为排序输出类型。将 15 毫升管架放入分拣室中。打开板电压并将板电压设置为大约 4， 000 伏。接下来，通过选择流设置按钮打开测试模式。调整充电相位滑块和偏转滑块，以确保流的图像与收集管对齐。然后选择复选标记按钮。在计算机上登录到 Summit 工作站。从文件主菜单创建新协议。然后，通过在 Summit 软件控制面板中选择直方图选项卡来创建点图。选择前向散射作为 x 轴参数，选择侧散射作为 y 轴参数。然后右键单击工作区中新点图的轴，并以对数形式显示前向散射和侧散射。选择采集选项卡并找到采集参数面板。启用启用参数子菜单中的所有信号。选择前向散射作为触发参数，并将阈值设置为0.01。将前向散射和侧向散射的电压和增益调整为 250 和 0.6，以确保前向散射图与侧向散射图中的事件和群体分开。通过将一微升微球加入一毫升双蒸水中，将荧光聚苯乙烯微球稀释成 100、200 和 300 纳米悬浮液。然后依次加载微球悬浮液。在峰会软件的采集菜单下选择开始，记录20， 000个事件，记录事件后点击停止。右键单击前向散点图与侧散点点图以创建矩形并拖动以调整大小。通过右键单击这些区域来重新定位电子噪声和 100 纳米微球群的区域，将它们分别重命名为 R7 和 R4。重复上述步骤，依次用矩形框住200纳米和300纳米微球，并分别将其重命名为R5和R6。最后，根据电子噪声和200纳米粒子的位置确定50至200纳米的分选区域，将其定义为R8。在排序逻辑和统计信息面板中编辑排序决策，方法是双击左侧流或 L1 流的空白字段，然后选择名为 R8 的区域以创建排序逻辑。选择中止纯度模式，并为L1流选择高于一滴的液滴包络。加载先前制备的细胞外囊泡或EV混合物的500微升样品。按 Summit 中采集菜单下的“开始”按钮获取数据，然后单击“停止”。接下来，按排序菜单中的开始，将 50 至 200 纳米大小的小细胞外囊泡或 sEV 收集到 15 毫升离心管中。对标准尺寸的聚苯乙烯微球进行流式细胞术分析，以定位前向散射与侧向散射图中的50至200纳米尺寸范围，揭示了可区分的颗粒信号。R4、R5 和 R6 分别指 100、200 和 300 纳米颗粒的位置。R7是电子噪声作为50纳米颗粒的检测限，R8表示50至200纳米颗粒的位置范围。通过纳米颗粒跟踪分析（NTA）从胰腺一细胞中提取的EVs混合物的粒度分布在超速离心后的40至400纳米的宽范围内。进行流式细胞术分选以分离和纯化R8区域内的50至200纳米sEV。通过NTA验证了分离质量，发现分选后的sEV粒径范围在50-200纳米之间。通过TEM进一步观察到sEV的存在，并通过蛋白质印迹分析确认分离的sEV含有CD6，CD63和CD81的标志物。该方法允许分离小细胞外囊泡和小细胞外囊泡基因表达的分类或专业分析，开辟了许多下游研究应用。

Research

JoVE Journal

Biochemistry

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

科研

生物化学

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Isolation of Tissue Extracellular Vesicles from the Liver

组织细胞外囊泡与肝脏分离

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in ...

Imaging of Extracellular Vesicles by Atomic Force Microscopy

原子力显微镜对细胞外囊泡的成像

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter

使用多柱板适配器的经济性和多功能性高通量蛋白质净化系统

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

High-Throughput Screening for Protein Crystallization

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography (Video) | JoVE 

高通量筛选以获得用于蛋白质晶体学的晶体命中

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into ...

Isolating Small Extracellular Vesicles and Extracting Their Peptidome

Identification of Peptides of Small Extracellular Vesicles from Bone Marrow-Derived Macrophages

Peptidome Extraction from Small Extracellular Vesicles Isolated from Bone Marrow-Derived Macrophages (Video) | JoVE

从骨髓来源的巨噬细胞中鉴定小细胞外囊泡的肽

Small extracellular vesicles (sEVs) are typically secreted by the exocytosis of multivesicular bodies (MVBs). These nanovesicles with a diameter of ...