Name: nanoparticle tracking analysis for the quantification and size determination of extracellular vesicles
Uploaded: 2021-03-28T14:00:00
Description: Watch this Scientific Journal Video about Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles at JoVE.com

这项工作得到了国家卫生研究院（ES030973-01A1，R01ES025225，R01DK066525，P30DK026687，P30DK063608）的支持。我们感谢霍里巴仪器公司博士杰弗里·博德科姆帮助校准仪器。

这些样本的所有工作均按照机构动物护理和使用委员会和机构审查委员会的准则进行。 图2描绘了NTA方法的示意图概述。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62447/62447fig02.jpg"> 图2：使用粒子跟踪仪器的NTA方法概述。 样品已准备并插入仪器。打开 NTA 软件，调整记录参数，并集中示例?...

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在这里，我们演示了电动汽车 NTA 的协议，以同时测量各种颗粒大小的大小分布，并测量多分散样品中的总 EV 浓度。在这项研究中，小鼠围产层脂肪组织和人类血浆被用作电动汽车的来源。然而，从其他组织或生物液体（如血清、尿液、唾液、母乳、羊水和细胞培养超高纳特）中分离出来的EV也可用于NTA。聚苯乙烯珠标准的测量确保了仪器得到适当校准，并证明该 NTA 仪器能够正确测量纳米粒子的...

细胞外囊泡 （EV） 是小 （0.03-2 μm） 膜绑定囊泡分泌几乎所有细胞类型1.它们通常被称为"外显子"、"微囊"或"凋形体"，这取决于它们的释放机制和大小2。虽然最初认为电动汽车只是消除细胞废物以维持平衡3的一种手段，但现在我们知道，它们也可以通过分子物质的转移参与细胞际交流，包括DNA、RNA（mRNA、microRNA）、脂质和蛋白质4、5，它们是正常生理学和病理过程的重要调节器。 5，6，7，8。
有许多不同的方法来隔离和量化电动汽车，这在其他地方已经描述9，10，11，12。使用的隔离协议以及电动汽车的来源可以极大地影响电动汽车的产量和纯度。即使是差异离心，长期被认为是外体隔离的"黄金标准"方法，也可能受到巨大的变异性，随后影响获得的EV种群和下游分析13。因此，EV隔离和量化的各种不同方法使得比较、复制和解释文献14中报道的实验结果变得困难。此外，EV 释放可以由细胞条件或各种外部因素调节。研究表明，电动汽车通过保护细胞免受细胞内应激15在维持细胞平衡方面发挥作用，因为一些研究表明，细胞压力会刺激EV分泌。例如，在细胞接触缺氧、内质视网膜应激、氧化应激、机械应激、香烟烟雾提取物和颗粒物空气污染16、17、18、19、20、21、22之后，EV释放量增加。EV 版本也已显示在体内进行了修改：小鼠受高脂肪饮食或禁食16小时释放更多的脂肪细胞EV23。要调查特定的治疗或条件是否改变 EV 释放，必须准确确定电动汽车的数量。对EV尺寸分布的评估也可能表明电动汽车的主要亚细胞起源（例如，晚期内分泌体/多血管体与等离子体膜的融合与等离子膜的萌芽）24。因此，需要采用强有力的方法来准确测量正在研究的电动汽车准备的总浓度和尺寸分布。
纳米粒子跟踪分析 （NTA） 是解决方案中电动汽车可视化和定性快速和高度敏感的方法。详细解释这种方法的原则，并与评估EV尺寸和浓度的替代方法进行比较，先前已描述25，26，27，28。简言之，在 NTA 测量期间，当电动汽车被激光束照射时，它们会被散射的光线所可视化。散射光通过显微镜聚焦在记录粒子运动的相机上。NTA 软件跟踪每个粒子的随机热运动（称为布朗运动），以确定扩散系数，该扩散系数用于使用斯托克斯-爱因斯坦方程计算每个粒子的大小。NTA于2011年首次应用于生物样本中的电动汽车测量。直到最近，只有两家主流公司提供商用 NTA 仪器29，直到采用 ViewSizer 3000（以下简称粒子跟踪仪器），它使用新型硬件和软件解决方案的组合来克服其他 NTA 技术的巨大局限性。
粒子跟踪仪器通过分析液体样品中的纳米粒子，通过分析其布朗运动来描述纳米粒子的特征，并通过分析引力沉降来描述较大的微米大小的粒子。该仪器独特的光学系统包括三个激光光源（450纳米、520纳米和635纳米）的多光谱照明，使研究人员能够同时分析各种粒子大小（例如外显子、微囊）。图 1显示了仪器设置的示意图。
在这里，我们演示了如何使用新型的 NTA 仪器对孤立的鼠标和人类 EV 进行颗粒大小分布和浓度测量。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62447/62447fig01.jpg"> 图1：粒子跟踪仪器光学系统。 NTA 仪器使用以下波长为 450 nm、520 nm、635 nm 的三种激光照明粒子。从单个粒子中检测和跟踪分散光的视频记录，由面向 90° 的数字摄像机从 cuvette 检测和跟踪。 <a href="https://www.jove.com/files/ftp_upload/62447/62447fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1X dPBS</td>
			<td>VWR</td>
			<td>02-0119-1000</td>
			<td>To dilute samples</td>
		</tr>
		<tr>
			<td>100 nm bead standard</td>
			<td>Thermo Scientific</td>
			<td>3100A</td>
			<td>To test ViewSizer 3000 calibration</td>
		</tr>
		<tr>
			<td>400 nm bead standard</td>
			<td>Thermo Scientific</td>
			<td>3400A</td>
			<td>To test ViewSizer 3000 calibration</td>
		</tr>
		<tr>
			<td>Centrifugal Filter Unit</td>
			<td>Amicon</td>
			<td>UFC901024</td>
			<td>To filter PBS diluent</td>
		</tr>
		<tr>
			<td>Collection tubes, 2 mL</td>
			<td>Qiagen</td>
			<td>19201</td>
			<td>For isolation of human plasma extracellular vesicles</td>
		</tr>
		<tr>
			<td>Compressed air duster</td>
			<td>DustOff</td>
			<td>DPSJB-12</td>
			<td>To clean cuvettes</td>
		</tr>
		<tr>
			<td>Cuvette insert</td>
			<td>HORIBA Scientific</td>
			<td>-</td>
			<td>Provided with purchase of ViewSizer 3000</td>
		</tr>
		<tr>
			<td>Cuvette jig</td>
			<td>HORIBA Scientific</td>
			<td>-</td>
			<td>To align magnetic stir bar while placing inserts inside cuvette; Provided with purchase of ViewSizer 3000</td>
		</tr>
		<tr>
			<td>De-ionized water</td>
			<td>VWR</td>
			<td>02-0201-1000</td>
			<td>To clean cuvettes</td>
		</tr>
		<tr>
			<td>Desktop computer with monitor, keyboard, mouse, and all necessary cables</td>
			<td>Dell</td>
			<td>-</td>
			<td>Provided with purchase of ViewSizer 3000</td>
		</tr>
		<tr>
			<td>Ethanol (70-100%)</td>
			<td>Millipore Sigma</td>
			<td>-</td>
			<td>To clean cuvettes</td>
		</tr>
		<tr>
			<td>ExoQuick ULTRA</td>
			<td>System Biosciences</td>
			<td>EQULTRA-20A-1</td>
			<td>For isolation of human plasma extracellular vesicles</td>
		</tr>
		<tr>
			<td>Glass scintillation vials with lids</td>
			<td>Thermo Scientific</td>
			<td>B780020</td>
			<td>To clean cuvettes</td>
		</tr>
		<tr>
			<td>&#34;Hook&#34; tool</td>
			<td>Excelta</td>
			<td>-</td>
			<td>Provided with purchase of ViewSizer 3000</td>
		</tr>
		<tr>
			<td>Lint-free microfiber cloth</td>
			<td>Texwipe</td>
			<td>TX629</td>
			<td>To clean cuvettes and cover work surface</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes, 2 mL</td>
			<td>Eppendorf</td>
			<td>22363344</td>
			<td>For isolation of human plasma extracellular vesicles</td>
		</tr>
		<tr>
			<td>Stir bar</td>
			<td>Sp Scienceware</td>
			<td>F37119-0005</td>
			<td></td>
		</tr>
		<tr>
			<td>Suprasil Quartz cuvette with cap</td>
			<td>Agilent Technologies</td>
			<td>AG1000-0544</td>
			<td>Initially provided with purchase of ViewSizer 3000</td>
		</tr>
		<tr>
			<td>ViewSizer 3000</td>
			<td>HORIBA Scientific</td>
			<td>-</td>
			<td>Nanoparticle tracking instrument</td>
		</tr>
	</tbody>
</table>

nanoparticle tracking analysis for the quantification and size determination of extracellular vesicles

细胞外囊泡（EV）的生理和病理生理作用已日益得到认可，使EV领域成为一个快速发展的研究领域。电动汽车隔离有许多不同的方法，每个方法都有明显的优缺点，影响电动汽车的下游产量和纯度。因此，通过选定的方法将EV准备从给定来源中分离出来，对于解释下游结果和比较实验室的结果非常重要。有各种方法来确定电动汽车的大小和数量，这些方法可以根据疾病状态或根据外部条件进行更改。纳米粒子跟踪分析 （NTA） 是用于单个 EV 的高通量分析的突出技术之一。在这里，我们提出了一个详细的协议，量化和大小确定EV从小鼠围产儿脂肪组织和人类血浆使用NTA的突破性技术，代表该领域的重大进展。结果表明，该方法可以提供可重复和有效的总粒子浓度和大小分布数据，用于使用不同方法从不同来源分离的电动汽车，如传输电子显微镜所证实的。该仪器的NTA改造将解决NTA方法标准化的需要，以提高电动汽车研究的严谨性和可重复性。

在此演示之前，首先测试了仪器的校准，通过测量聚苯乙烯珠标准的大小分布来确保获得数据的有效性。我们使用默认记录参数和本协议中建议的处理设置（图 8）测试了 100 nm 和 400 nm 珠的大小分布。
对于 100 nm 聚苯乙烯珠标准，测量了 4.205 x 107 颗粒/mL 的浓度。平均值（标准偏差，SD）大小为102（±17）nm，变异系数（CV）为0.16。D10/D50/D90 值为 76...

Watch this Scientific Journal Video about Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles at JoVE.com

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

我们演示了如何使用新型纳米粒子跟踪分析仪器来估计从小鼠围产层组织和人类血浆中分离出来的细胞外囊泡的大小分布和总颗粒浓度。

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 ...

NTA 结果非常容易出现操作员偏差。此协议演示了更改 NTA 参数对已获得的结果的影响。标准化方法将有助于提高 NTA 分析的严谨性和可重复性。分析 cuvette 中的样本允许在每个视频中捕获统计随机样本。这可产生更可重复的数据和各种尺寸的粒子可视化。由于在推荐的粒子浓度范围内获取样品可能很困难，因此一定要进行连续稀释，以确定理想的稀释因子。安东尼·费兰特实验室的博士生蔡坤恒将演示这一程序。要为纳米粒子跟踪分析做好准备，请使用无绒毛材料覆盖工作空间，以防止纤维进入幼崽。戴着手套，将装有搅拌棒的夹具放在磁性小插曲上。使用钩形工具将插入插入到库维特中，插入的插口在库维特前面可见。使用移液器将 400 到 500 微升纯化的细胞外囊塞慢慢添加到切口上，通过插入孔，通过轻轻的管道混合样品，而不引入气泡，然后盖住小面包，根据需要敲出气泡，并使用无绒布擦拭切口的外部表面。要分析稀释剂或样品的颗粒浓度，请打开计算机工作站和仪器，启动粒子跟踪分析程序。提示时，单击 NTA 并打开录制选项卡。按照屏幕上的说明填写所有必要的示例信息。对于 EV 跟踪分析，将稀释剂设置为 PBS。盐度将自动填充到 9% 为了获得稀释物或样品颗粒浓度，请打开仪器盖并取下将 cuvette 放置位置的保护盖。将 cuvette 以正确的方向加载到仪器中，插入器的插件朝面向摄像机，并更换盖子和仪表盖。单击流箭头打开相机并单击雪佛龙箭头以扩展记录设置。调整对焦，直到相对较小的颗粒清晰可见。要设置小 EV 量化分析，将帧速率设置为每秒 30 帧，曝光时间设置为 15 毫秒，搅拌时间设置为 5 秒，等待时间设置为 3 秒，蓝色、绿色和红色激光动力分别设定为 210、12 和 8 毫瓦，每个视频的帧数设置为 300，增益到 30 分贝。调整对焦，直到相对较小的颗粒清晰可见。增加变焦和/或增益有助于粒子对焦。但是，如果您增加增益，请记住在录制前将其设置为 30 分贝。一旦粒子处于焦点状态，将变焦设置设置设置为 0.5 倍，以节省带宽并防止丢失帧并单击记录以开始录制视频。当提示显示已录制视频时，单击"确定"以完成录制并选择过程选项卡。如果在录制时任何视频中都可以看到非常大的颗粒，请导航到录制的视频目录，并在处理之前删除任何有问题的视频。检查禁用音频检测覆盖框，并将功能直径设置为 30。单击过程以启动视频处理并查看实时分布图。处理完成后，单击"确定"并选择绘图选项卡。对于电动汽车，将主图表显示为日志箱二氧化硅。图形的其他功能，如更改 x 轴以设置所生成图形的集成区域，可以自定义。要创建结果的 PDF 报告，请单击报告按钮。将显示因稀释因子和分布宽度的平均值、中位数、模式大小和浓度调整。在任何样品之前应执行二分红的 NTA，以便从 EV 样品颗粒浓度中减去此空白的浓度。要在分析后清洁幼崽，清空幼虫，用去离子水将幼崽完全填满 10 到 15 次，用 70 到 100% 乙醇将三次切除任何残留样品。用无绒毛纤维布干燥幼虫的外部，用压缩空气除尘器干燥内部。要清洁插入物和搅拌条，将材料放入含有 70 至 100% 乙醇的玻璃闪烁小瓶中，并大力摇动小瓶。然后冲洗插入物，在去离子水中搅拌棒，如所示，摇晃，然后用无绒布将其晾干。干燥后，立即将所有清洁部件放入存储，直到下一次分析。在进行分析之前，使用聚苯乙烯珠测试仪器校准，以确保获得的数据的有效性。据观察，粒子跟踪仪器准确地报告了100纳米的马诺分散珠的大小，但只密切报告了400纳米珠子的大小。因此，此协议的仪器设置对于体积接近 100 纳米的较小粒子更准确。使用这些设置，报告的粒子浓度相应地与稀释因子相符，表明仪器能够在各种稀释时准确检测粒子浓度，而技术复制器之间变化不大。每毫升小鼠组织衍生EV样本中4.41倍10至10个粒子的最佳稀释量被确定为1000至3000粒。在此分析中，增加增益可提高相机的灵敏度，从而增加更小颗粒的可视化性。将蓝色激光功率从 70 毫瓦增加到 210 毫瓦，同时保持绿色和红色激光功率不变，将报告的平均粒子大小从 122 纳米增加到 105 纳米，并将报告的总粒子浓度从 1.1 倍 10 增加到第八倍，从 10 倍增加到第八倍。增加红色激光的功率使报告的平均粒子大小从175纳米增加到246纳米，并降低了报告的总粒子浓度。增加绿色激光功率导致报告的平均粒子大小减少，报告的总粒子浓度增加。找到合适的稀释，将样品置于最佳检测范围内，可能需要为每个样品尝试几次。库维特清洁还需要格外小心的处理。我们建议在EV粒子大小和浓度测量中应用多种正交方法。动态光散射、电阻脉冲传感、传输电子显微镜和单粒子干涉反射成像感应也可以对电动汽车进行特征化。

Research

JoVE Journal

Biology

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

正确的蛋白浓度的动力学结构与功能分析和亲和力测定的重要性

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

科研

生物学

An Innovative Method for Exosome Quantification and Size Measurement

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 ...

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

快速，特异微孔板检测为内和细胞外抗坏血酸在培养细胞的测定

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within ...

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 ...

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 ...

Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes

用于荧光标记大促卵体定量的自动成像和分析

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and ...

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

通过共聚焦显微镜成像分析进行细胞外囊泡摄取测定

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular ...

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

三维细胞外囊泡的直接随机光学重建显微镜

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often ...

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 ...