The authors would like to thank Christina Ball&#225;zs, Hug Aubin and J&#246;rn H&#252;lsmann for the critical reading of the manuscript and excellent editorial assistance. Moreover, the authors thank Gisela Mueller for the technical assistance. The authors thank Particle Metrix GmbH for providing the funds covering the publication costs.

注：在这项工作中提出的试验已获得杜塞尔多夫大学的机构伦理委员会。 1.外来体的制备<ol><li>通过静脉穿刺采集全血中3柠檬酸管（总共9毫升）中。倾血液入15ml Falcon管中。 </li><li>离心样品在1500×g离心20分钟，在4℃下，从等离子体引发的细胞的分离。上清转移到一个新的15毫升猎鹰管。 </li><li>离心样品在2800×g离心20分钟，在4℃下，以从血浆中除去所有的细胞（无细胞的血浆; CFP）。转移CFP超离心管，每管1毫升。 </li><li>离心机以100,000×g离心90分钟，在4℃下以耗尽外来体。取出900微升上清液。重悬浮沉淀在超速离心管中的剩余的100微升。添加900微升PBS。 </li><li>离心再次以100,000×g离心30分钟，在4℃。取出900微升苏pernatant。重悬浮沉淀与剩余的100微升。 </li><li>转印在40ml蒸馏水洗涤5到20微升的重新悬浮液。通过450nm的过滤器过滤该悬浮液以外来体从较大的颗粒中分离出来。使用这个最终的悬浮液粒子的测量。 </li></ol>粒子跟踪仪器2.启动程序<ol><li>开始通过双击该软件图标的程序。点击各种软件的选项卡（“细胞检查”，“测量”，“分析”），它们之间在整个协议的切换。 </li><li>按照屏幕上的说明启动过程的自动化实现。选择框两种细胞的质量检查和自动对齐。 注：以上任一步骤可以单独在必要时可重复按下“电池检查”选项卡上的按钮（A）或（B）。 </li><li>打开NTA仪器的入口和出口，并注入10米升蒸馏水，用注射器入通过进气口的小区的信道。关闭出口孔作为水的最后量被注入。将在出口的烧杯收集废液。确保测量单元是不含气泡的，并且不注射器气泡进入系统。立即关闭进气口。 </li><li>点击“OK”执行小区质量检查。几秒钟后，该软件会显示电池质量的结果。 </li><li>如果有任何颗粒仍可视化软件的实时取景画面，或如果质量检查的结果是唯一的好或差，重复步骤2.3，直到剩下的颗粒从测量单元中删除。每次测量后，也重复步骤2.3，以避免粒子的聚集。如果重复蒸馏水注射细胞检查不会产生一个“好”的结果，进行2.9。 </li><li>制备含统一200纳米大小的POL控制悬架要使用ystyrene颗粒对准激光器和显微镜的焦点。添加一滴悬浮液浓缩物，由仪器制造商提供的，500毫升蒸馏水，以获得所需的浓度，使得600±100颗粒被在实时取景显示的每个画面。 </li><li>注入对准悬浮到NTA仪器如在步骤2.3中所述。按“OK”，开始自动校准，自动的例行通过该系统会自动找到两个焦点的最佳位置。 </li><li>当出现提示，说明该系统现在准备的实验，点击确定开始测量。 </li><li>偶尔，由冲洗细胞用乙醇30％的溶液手动清洁实验之间的小区的信道。清洁时，软件会显示一个自动错误报告的通道。 </li></ol> 3.测定样品的<ol><li>同花顺前EA蒸馏水通道CH样品测量（如在步骤2.3中所述）。 </li><li>注入的外来体悬浮液中的信道（其制备在第1），如在步骤2.3中所述。 </li><li>根据需要调整软件中的以下主要参数： <ol><li>灵敏度。为了找到最优灵敏度范围，按一下按钮“粒子与灵敏度的数量”，以显示一个曲线为每个画面测量的颗粒为不同的灵敏度水平。选择了该曲线的最大斜率前一个灵敏度水平。更高的灵敏度级别允许更多的小颗粒的可视化，但也增加了相关的背景噪音的问题。 </li><li>最低亮度。选择了20的切体测量使用此功能作为过滤器起始亮度。调整这个参数多达空白出强烈的光散射粒子。规范下来放大弱散射的文物。变化的亮度根据需要在整个实验。 注：粒子轻散射太强烈重叠并且可以排除由错误的分析。 </li><li>最小和最大尺寸。设置数字滤波器通过调节的最小和最大尺寸，以消除从过大的颗粒像素噪声和无用飞散。使用范围为10至500个像素为外来的测量。 </li><li>快门。调整的时间，该照相机是打开以1/30​​0秒的周期。 </li></ol></li><li>住读出参数： <ol><li>通过点击“实时图像”按钮，数字和模拟视图手法在任何点之间切换。 </li><li>在整个实验中，监测散射强度栏，其中显示的样本作为颜色编码表示的饱和度状态。不分析，如果外来体散射条为红色。在这样的情况下进一步稀释样品，以避免这种现象。如果在实验条件禁止的操作的样品悬浮液，下调的敏感性或上调在软件 - S的亮度ettings改善测量结果。 注意：当具有非常高浓度的颗粒的样品进行分析的散射会熔合单个颗粒和它们被计为一个单颗粒。 </li><li>注意从显示在视野计数的粒子数。 </li></ol></li><li>点击“测量”选项卡上。 </li><li>选择在“运行选项”阵列设置。 <ol><li>每次收购之前，选择一个反复检查颗粒漂移测试“检查运动”。在开始整体分析之前进行测试至少一次。如果漂移是高于20微米/秒，持续测量使得样品停止流动之前等待。 </li><li>选择的实验（5）的数量和它们之间的时间延迟（0）。 </li><li>进行“抽样检查”，如果必要的。这个测试是自动对准在启动过程的一部分，并且可以重复之后根据需要。 </LI&gt; </ol></li><li>最后点击“运行视频采集”按钮。在新窗口中，定义周期（15）的数量和测量位置的数目（11）。 注：激光头，并在显微镜可以被移动到分析的粒子数在11个不同的位置上。根据实验的需求，决定是否在实验应该在一个单一位置，或在不同的位置上进行。 <ol><li>确认的最小持续时间的颗粒具有通过设置视频分辨率（低）被跟踪要被计数为一个单独的粒子。较低的分辨率结果在更短的时间跟踪。 </li><li>选择一个文件名，然后单击“确定”开始测量。 </li></ol></li></ol> 4.解读结果<ol><li>查看测量后显示下列结果和参数中的“结果”标签：跟踪粒子（1）的总数目，的部分（2）的平均数目每位置，和（3）颗粒浓度icles。 <ol><li>查看结果平均表粒径（直径，表面和体积）的颗粒数的百分比的数值比，以及平均和标准分化的值。 </li><li>如果存在于图形分析多于一个峰使用峰分析表。此表显示的颗粒，比第一或分别第二峰值较小的分布。 </li></ol></li><li>查看测量后计算看到颗粒通过尺寸分布的曲线图。使用在显示模式和图表上方的图标来改变设置。 </li></ol>

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This work was supported by the institutional funds of the Dept. of Cardiovascular Surgery, Medical Faculty, HHU. The publication costs of this study were provided by Particle MetrixGmbH.

我们证明用于从血液和本纳米粒跟踪外来体分离为一个新颖的和创新的方法来测量在生物流体外来体的大小和浓度的详细协议。在所提出的实验人外周血用作外来体的来源。然而，其他来源，如尿，痰，细胞培养上清等，也可以被用来作为测试材料。 基于在人类中外来体的浓度的生物变异性，从不同的个体测定可设有外泌体的一个显着变化的浓度。然而，在生物测试探针粒子的浓度可能对结果产生影响。因此，需要一种用于探针的稀释的可靠和标准化的方法。在该方法包含外来体血浆9毫升外周血全血中产生。使用差速离心步骤外来体丸粒从分离1毫升血浆并重新悬浮于5ml蒸馏水以产生悬浮外来体的工作示例。此预先定义的设置为我们提供了颗粒的适宜浓度， 即 NTA在适当的散射强度。旁边的体积和稀释的方面，所使用的溶液的组合物也是重要的。我们已经用蒸馏水进行最后再悬浮外来体。当然，也可以使用不同的介质进行最后稀释步骤，根据所述实验设置的要求。然而，在离子的溶液具有缓冲能力测试样品特别是当ζ电势测量的情况下，谨慎的稀释在紊流自由条件是有用的。对多个样品的直接比较，我们建议保持对所有样品相同的稀释程度。除了敏感性和视频分辨率都设置可以事后通过使用ZetaView分析软件来改变。 10等系统。我们相信，有关样品处理结果的可重复性和实用性方面是至关重要的选择方法序列切体的评价。我们还认为，理想的检测系统应该消除可能与测量结果干扰用户取决于因素。即这里介绍的外来体分析方法满足了容易处理，以高度的标准。作为另一个优点，在一个快速的过程中进行样品的半自动化分析，产生的结果在很短的时间。外来体的在线可视化辅助分析仪GA在外来体的特性， 比如，总浓度瞬间的想法。 一个非常简单的，但优雅除了这里介绍的测量技术是利用抗体标记的外来体和散射激光束通过在检测器的前面使用前的过滤器捕获。以这种方式外来体亚群可根据其表面抗原和其它生物的相关特征是技术上可访问的选择性荧光染色来区分。 我们的粒子跟踪仪器的当前版本的一个缺点是，在非常高的灵敏度水平工件诸如背景噪声可能成为本，这是基于该单元沟道壁。一个技术解决方案，并改善当前系统可能在不久的将来变得可用。通过在通道壁上的激光散射光的这种方法反射将被减少，从而增加了<html>测得的信号和获得的数据和降低检出限的准确性。 尽管当前使用的协议，用于外来体分离是公认的和所施加的粒子跟踪仪具有30nm的下部粒度分布的显着精确的分辨率，但不保证所检测的粒子确实完全正确的外来体。其他颗粒，如死细胞碎片或更大的蛋白质复合物也可以存在于外来体悬浮液，并导致假阳性信号。一个可靠的方法，使这种“污染”可排除可能是电子显微镜（EM），无论是传输EM或扫描EM。不幸的是，没有一般和特殊标记的外来已经确定，到目前为止，虽然有一些先前提出的表面标志已获得了越来越多的关注，其中包括四旋（TSPAN），CD81，C63，CD9 等 11。 “&gt;无论对外来体生物，特别是有关外来体特异性标记研究的未来进展，那就是这里介绍的协议将提供一个强有力的方法用于分离和检测外来体。浓度和尺寸可以很容易地在软件辅助确定直接的方式，该加的选择性荧光标记或荧光团偶联的抗体可能会进一步提高NTA的呈现的方法的可能的应用。

循环外来体的确切功能仍未知的很长一段时间。即使是现在外来体的完整路径机制尚不完全清楚。由于外来体携带的抗原，蛋白质和涉及到他们的亲本来源的细胞，它们的功能的细胞 - 细胞信号发送器的RNA（mRNA和miRNA）的主要被给予优先权。 许多不同的方法已经在文献中描述的用于分离和外来体1,2的定量检测。然而，在“金标准”没有达成共识已经达成。与此同时，大多数科学家活跃在切体研究领域的同意，孤立的一致方法非常必要的，以实现更高程度不同的报告和研究报告之间的可比性。 荧光激活细胞分选（FACS）是用于外来体分析3中最常见和普遍的工具。 FACS有奔EFIT即，通过荧光标记，来自不同来源的细胞可以在一个步骤中进行比较。 FACS的主要缺点是，该方法是不够敏感，以确定颗粒小于0.5微米4，而外来体一般是30-120纳米之间在直径为5，甚至更少来测量它们的大小。 扫描电子显微镜（SEM）和透射电子显微镜（TEM）的其他工具进行颗粒尺寸和外来体的形态学分析。但是，这两个SEM和TEM的缺点是样品的制备是费时，这两种方法涉及劳动密集的步骤和各具有工件生成的一些风险。既不方法适合于高样品通量和几千一个样品的单一颗粒的表征。此外，可以同时或至少分析在非常短的周期进行定量分析，为临床日常其中样品具有通常被难以进行。新一代的技术，现在让我们来分析外来体，恕不另行紧张的筹备工作（ 例如，环境SEM）。这些现代化的技术仍然相当不方便分析含外来体，以确定它们的平均数量和规模分布6大体积悬浮液。 另一个可视化和外来体的分析高度敏感的方法是纳米粒子跟踪分析（NTA）。这种方法利用了物理两种不同的原则。首先，颗粒通过散射当它们被照射激光束的光检测。第二个现象被称为布朗运动，根据该不同的颗粒在液体中的悬浮液的扩散成反比它们的大小。在后一种情况下，该运动也取决于温度和液体的粘度。然而，这样的速度是直接关系到颗粒大小和所使用的NTA。使用软洁具为基础的分析，散射光从单颗粒数字图像记录。散射光斑点的图解和其运动速度提供便利的总粒子数和粒径分布的测定数据。这种技术是对小于100纳米的平均直径的粒子分析特别强大。 大小和浓度测量与ZetaView布朗和电泳运动视频分析显微镜进行。这是一种半自动化的桌面纳米颗粒分析仪对液体样品（以下简称为颗粒跟踪仪）。它由粒子追踪分析仪，以及与用于数据分析的软件的笔记本电脑。异质生物样品是作为适合于这种方法，无机颗粒的更均匀的悬浮液。一种激光散射显微镜用摄像机被用于颗粒的检测和用于OBSErvation他们的行动。而在显微镜轴线是水平的，并聚焦到填充有含有外来体的悬浮液的小区的信道时，激光束是垂直取向的。通过激光散射的光，这是根据90°经由显微镜（ 图1）所记录的数字视频相机照射的粒子。的散射光的强度可以观察粒子大60纳米的直径。在这样一种设置的粒子的亮度不粒度的唯一指示。当没有施加电场，粒子运动只有如下布朗运动并且可以用作用于计算粒径的指标。然而，该仪器还能够在整个小区通道施加电场的。当进行这一领域的潜力，极性和离子电荷的悬浮外来体的级成为他们移动的方向的进一步的决定因素。速度和方向，导致电泳莫相容性直方图。 同时发现，分析分离的外泌体的最佳方法是一个问题，另一个位于从不同的媒体，如血液，腹水，尿，乳汁，羊水或细胞培养基的外来体的有效的隔离。不同的方法已被描述迄今，其基于超速离心1，工业分离试剂（如Exoquick）7，磁珠抗原采用分离8或超滤步骤9。 在这个协议中，我们展示的切体隔离，通过超速离心的全过程，并展示了如何通过分析粒子跟踪仪含停牌产生的切体。提供特定的考虑因素的人血浆或细胞培养基衍生的外来体的分析。

<table border="0" cellpadding="0" cellspacing="0" width="645">
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			<td height="20" style="height:20px;width:159px;">Name of the Reagent</td>
			<td style="width:116px;"></td>
			<td style="width:124px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Citrate tube</td>
			<td>BD</td>
			<td>364305</td>
			<td>BD Vacutainer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Distilled water</td>
			<td>Braun</td>
			<td>3880087</td>
			<td>Aqua ad iniectabilia</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Falcon tube</td>
			<td>Greiner Bio One</td>
			<td>188271</td>
			<td>PP Tube, Steril 15ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultracentrifugation tube</td>
			<td>Beckman</td>
			<td>357448</td>
			<td>Microfuge Tube Polyallomer 1,5ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polybead</td>
			<td>Polysciences, Inc.</td>
			<td>07304</td>
			<td>2,6% Solids-Latex 
			Alignment Solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe (Filter)</td>
			<td>Braun</td>
			<td>4617053V</td>
			<td>5ml</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Syringe (ZetaView)</td>
			<td>Braun</td>
			<td>4606051V</td>
			<td>5ml</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Needle</td>
			<td>BD</td>
			<td>305180</td>
			<td>BD Blunt Fill Needle</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Filter</td>
			<td>Sartorius Stedim</td>
			<td>16555</td>
			<td>Syringe filter, hydrophilic, 450&#181;m</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Material Name&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultracentrifuge</td>
			<td>Beckman</td>
			<td>L8-M</td>
			<td>Rotor: 70Ti Ser. No E21078</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ZetaView</td>
			<td>Particle Metrix</td>
			<td>PMX 100, Type 101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5804R</td>
			<td>Rotor: A-4-44</td>
		</tr>
	</tbody>
</table>

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).

用于此示范试样示出了用于测量的85％的灵敏度，所述灵敏度曲线的最大斜率之前（ 图2）的最佳设置。亮度，最小/被选择最大值作为建议的协议。 5.3颗粒/ ml的浓度×10 6个，测定，而颗粒的平均粒径为0.149微米，其中大多数是0.137微米。 测量后收到的值可以保存在一份报告名为.pdf的文件格式或为.txt导出到数据库中。如在协议（第4节）中描述的图形可以调整为优选的。视频序列也被保存并且可以用于购买离线再分析。然而，在这样的离线分析，摄像机的事先取得设置不能追溯改变。 为了找到最佳参数设置用于测量中，我们在这里描述第对100纳米的聚苯乙烯尺寸标准的例子，仪器设置E优化。的两个参数，灵敏度和最大/最小尺寸上的视频图像和粒度分布的影响进行了详细的讨论。所有其他参数总结在表1中 。 的灵敏度（从50至94）上的模拟和数字图像的影响的视觉印象被可视化在图3中，从图像导出定量信息显示在图4中 ，基于表1中的设置，闵尺寸= 5和最大尺寸= 200检测到的颗粒相对于灵敏度的数目的典型关系示于图4A中 。在50和90中，检测到的颗粒的数量增加，灵敏度和急剧上升为灵敏度&gt; 90。 66和86（A）之间发现敏感性的最佳范围。与DIF得到粒度分布同的灵敏度设置如图4B。的粒度分布代表三个独立测量的平均值。对于过低的灵敏度（灵敏度= 62，红色曲线），只有少数粒子进行分析从而导致比较差的统计数据。分析颗粒的数目增加，灵敏度达到70（黄色曲线）和86（棕褐色曲线）之间的最佳。进一步增加了灵敏度引线与颗粒滴和粒度分布朝着更小的尺寸（灵敏度= 94，蓝色曲线）移位的数量粒度分布的恶化。 图4C示出的数量基于X50直径的趋势（50％颗粒比这更小的直径），为灵敏度的函数。在米色间隔，粒径的相对标准偏差小于8％，并对应于最佳间隔A中的红色区域表示RSD&gt; 8％为差统计（灵敏度太低）或宽DISTRI的结果butions与移位到更小的尺寸（灵敏度太高）。 闵尺寸与最大尺寸的设置是应用于数字图像，以便与光斑尺寸小于最小尺寸小，比最大尺寸更大去除颗粒过滤器。由于散射光的能力，一个粒子产生一定大小的一个数字图像的一个点。光点的大小被测量为像素数（像素）。当粒子散射光非常好（ 例如，颗粒&gt; 200纳米或聚集体），光点尺寸是相当大的， 例如，&gt; 500像素。光点尺寸是相当小的（ 例如，&lt;10像素）取决于颗粒材料的小颗粒（ 例如，&lt;20纳米）。光斑大小（像素）可以不互换粒径（nm），因为它们是不相同的，并在这两个变量之间没有直接关系。的最小和最大尺寸的优化，用户可以过滤掉不需要的对象，如结块（最大尺寸）或小的对象，如背景噪声（最小尺寸）。对100nm的大小标准品的粒度分布最小/最大尺寸的影响示于图4D（灵敏度= 82）。当的时间间隔被设定为小的光斑尺寸（ 例如，最小= 1，最大值= 52;橙曲线），所分析的微粒的数目被减少，并且基于数X50直径略微移向较小的尺寸。的设置对于较大的斑点（最小= 40，上限= 1000;红色曲线）的结果是宽的粒度分布转向较大尺寸。为了获得相等的总数目的颗粒，这两个橙色和红色的分布的间隔的边界进行了调整，以匹配80的颗粒。与最佳设置的分布（分= 5，最大= 200;棕褐色曲线）由360粒。 进行了一系列成功的实验与外来体分离超速离心，并使用所提出的系统由NTA测量。所得到的数据是高度一致并确认再现性的高的水平。其他分离方法应该显示类似的结果。然而，稀释步骤被认定​​为特别关键的步骤和对所算出的总数目颗粒的影响，必须重新评估。 <img alt="图1" src="/files/ftp_upload/50974/50974fig1.jpg" /> 图1.原理图NTA的安装程序 。显微镜/视频轴和激光光束被正交定向为彼此交叉处的单元信道的横截面。光散射粒子会显示在软件的“实时视图”窗口。 <a href="https://www.jove.com/files/ftp_upload/50974/50974fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/50974/50974fig2.jpg" /> <stron颗粒相对于灵敏度曲线克&gt;图2号。颗粒与灵敏度曲线的数量自动灵敏度扫描期间显示在一个时刻一个位置的颗粒。这个图形可视化用于确定用于初始测量的最佳的偏好。甲灵敏度值被选择之前图中的最大斜率。重要的是要记住，文物没有在本次测试实验中消除，会影响图是很重要的。 <a href="https://www.jove.com/files/ftp_upload/50974/50974fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/50974/50974fig3.jpg" /> 被显示在模拟和数字影像。在实时取景画面上可视颗粒的灵敏度的图3的影响为50至94的灵敏度为模拟（顶行）的D数字（下排）的意见。当灵敏度太低，只有少数的粒子检测（左）。在最佳灵敏度的粒子显示为单一的点彼此（中）以及隔离。在相对 ​​高灵敏度的粒子融合在一起，导致图像质量差（右）。 <a href="https://www.jove.com/files/ftp_upload/50974/50974fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/50974/50974fig4.jpg" /> 图4.影响的敏感性最小和最大大小设置为控制100纳米的聚苯乙烯颗粒的样品（A）的敏感性与颗粒的检测数量的情节。最佳间隔是从66至86，前曲线的最大斜率。与几个灵敏度设置（62至94得到的（B）的粒度分布）;图表为过低（62）或太高（94）的灵敏度不捕获为100纳米的聚苯乙烯对照样品的粒度分布。 （C）的数50倍口径与敏感性; X50在米色间隔误差小于8％，最佳的间隔从66到最小值和最大值的粒度分布大小86（D）的影响;最佳参数（分= 5，最大值= 200）捕捉到正确的分布对照样品。 <a href="https://www.jove.com/files/ftp_upload/50974/50974fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td colspan="2"> 收购前的参数 </td></tr><tr><td>灵敏度</td><td>变量</td></tr><tr><td>快门</td><td> 40 </td></tr><tr><td>帧速率</td><td>每秒30帧</td></tr><tr><td>决议案</td><td>你好GH </td></tr><tr><td>周期</td><td> 10 </td></tr><tr><td>多个收购</td><td> 3 </td></tr><tr><td>位置</td><td> 1 </td></tr><tr><td colspan="2"> 收购后的参数 </td></tr><tr><td>闵亮度</td><td>三十</td></tr><tr><td>最大尺寸</td><td>变量</td></tr><tr><td>闵私迮</td><td>变量</td></tr></tbody></table> 表1的前和后采集参数为粒子跟踪仪器的设定综述。

Watch this Scientific Journal Video about An Innovative Method for Exosome Quantification and Size Measurement at JoVE.com

An Innovative Method for Exosome Quantification and Size Measurement

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 ...

an-innovative-method-for-exosome-quantification-and-size-measurement

Research

JoVE Journal

Biology

30.6K 次观看.  Heinrich-Heine-University Dusseldorf. 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.

视频: An Innovative Method for Exosome Quantification and Size Measurement

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

病毒融合动力学测量方法在单粒子能级

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

科研

生物学

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), and delta-cells (somatostatin) that are embedded in the exocrine tissues and comprise 1-2% of the entire pancreas. There is a close correlation between body and pancreas weight. Total beta-cell mass also increases proportionately to compensate for the demand for insulin in the body. What escapes this proportionate expansion is the size distribution of islets. Large animals such as humans share similar islet size distributions with mice, suggesting that this micro-organ has a certain size limit to be functional. The inability of large animal pancreata to generate proportionately larger islets is compensated for by an increase in the number of islets and by an increase in the proportion of larger islets in their overall islet size distribution. Furthermore, islets exhibit a striking plasticity in cellular composition and architecture among different species and also within the same species under various pathophysiological conditions. In the present study, we describe novel approaches for the analysis of biological image data in order to facilitate the automation of analytic processes, which allow for the analysis of large and heterogeneous data collections in the study of such dynamic biological processes and complex structures. Such studies have been hampered due to technical difficulties of unbiased sampling and generating large-scale data sets to precisely capture the complexity of biological processes of islet biology. Here we show methods to collect unbiased "representative" data within the limited availability of samples (or to minimize the sample collection) and the standard experimental settings, and to precisely analyze the complex three-dimensional structure of the islet. Computer-assisted automation allows for the collection and analysis of large-scale data sets and also assures unbiased interpretation of the data. Furthermore, the precise quantification of islet size distribution and spatial coordinates (i.e. X, Y, Z-positions) not only leads to an accurate visualization of pancreatic islet structure and composition, but also allows us to identify patterns during development and adaptation to altering conditions through mathematical modeling. The methods developed in this study are applicable to studies of many other systems and organisms as well.

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

计算机辅助大型胰岛质量，粒度分布和结构的可视化和量化

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), ...

Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method

Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large macromolecular complexes. Compared to X-ray crystallography, it has some unique advantages. First, single particle EM reconstruction does not need to crystallize the protein sample, which is the bottleneck in X-ray crystallography, especially for large macromolecular complexes. Secondly, it does not need large amounts of protein samples. Compared with milligrams of proteins necessary for crystallization, single particle EM reconstruction only needs several micro-liters of protein solution at nano-molar concentrations, using the negative staining EM method. However, despite a few macromolecular assemblies with high symmetry, single particle EM is limited at relatively low resolution (lower than 1 nm resolution) for many specimens especially those without symmetry. This technique is also limited by the size of the molecules under study, i.e. 100 kDa for negatively stained specimens and 300 kDa for frozen-hydrated specimens in general.
For a new sample of unknown structure, we generally use a heavy metal solution to embed the molecules by negative staining. The specimen is then examined in a transmission electron microscope to take two-dimensional (2D) micrographs of the molecules. Ideally, the protein molecules have a homogeneous 3D structure but exhibit different orientations in the micrographs. These micrographs are digitized and processed in computers as "single particles". Using two-dimensional alignment and classification techniques, homogenous molecules in the same views are clustered into classes. Their averages enhance the signal of the molecule's 2D shapes. After we assign the particles with the proper relative orientation (Euler angles), we will be able to reconstruct the 2D particle images into a 3D virtual volume.
In single particle 3D reconstruction, an essential step is to correctly assign the proper orientation of each single particle. There are several methods to assign the view for each particle, including the angular reconstitution1 and random conical tilt (RCT) method2. In this protocol, we describe our practice in getting the 3D reconstruction of yeast exosome complex using negative staining EM and RCT. It should be noted that our protocol of electron microscopy and image processing follows the basic principle of RCT but is not the only way to perform the method. We first describe how to embed the protein sample into a layer of Uranyl-Formate with a thickness comparable to the protein size, using a holey carbon grid covered with a layer of continuous thin carbon film. Then the specimen is inserted into a transmission electron microscope to collect untilted (0-degree) and tilted (55-degree) pairs of micrographs that will be used later for processing and obtaining an initial 3D model of the yeast exosome. To this end, we perform RCT and then refine the initial 3D model by using the projection matching refinement method3.

This article describes a standard method to get a three-dimensional (3D) reconstruction of biological macromolecules using negative staining electron microscopy (EM). In this protocol, we explain how to get the 3D structure of the Saccharomyces cerevisiae exosome complex at medium resolution using the random conical tilt reconstruction method (RCT).

单粒子电子显微镜外切体综合重建，使用随机的锥形倾斜方法

Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large ...

Tangential Flow Ultrafiltration: A  &ldquo;Green&rdquo; Method for the Size Selection and Concentration of Colloidal Silver Nanoparticles

Nowadays, AgNPs are extensively used in the manufacture of consumer products,1 water disinfectants,2 therapeutics,1, 3 and biomedical devices4 due to their powerful antimicrobial properties.3-6 These nanoparticle applications are strongly influenced by the AgNP size and aggregation state. Many challenges exist in the controlled fabrication7 and size-based isolation4,8 of unfunctionalized, homogenous AgNPs that are free from chemically aggressive capping/stabilizing agents or organic solvents.7-13 Limitations emerge from the toxicity of reagents, high costs or reduced efficiency of the AgNP synthesis or isolation methods (e.g., centrifugation, size-dependent solubility, size-exclusion chromatography, etc.).10,14-18 To overcome this, we recently showed that TFU permits greater control over the size, concentration and aggregation state of Creighton AgNPs (300 ml of 15.3 &mu;g ml-1 down to 10 ml of 198.7 &mu;g ml-1) than conventional methods of isolation such as ultracentrifugation.19
TFU is a recirculation method commonly used for the weight-based isolation of proteins, viruses and cells.20,21 Briefly, the liquid sample is passed through a series of hollow fiber membranes with pore size ranging from 1,000 kD to 10 kD. Smaller suspended or dissolved constituents in the sample will pass through the porous barrier together with the solvent (filtrate), while the larger constituents are retained (retentate). TFU may be considered a "green" method as it neither damages the sample nor requires additional solvent to eliminate toxic excess reagents and byproducts. Furthermore, TFU may be applied to a large variety of nanoparticles as both hydrophobic and hydrophilic filters are available.
The two main objectives of this study were: 1) to illustrate the experimental aspects of the TFU approach through an invited video experience and 2) to demonstrate the feasibility of the TFU method for larger volumes of colloidal nanoparticles and smaller volumes of retentate. First, unfuctionalized AgNPs (4 L, 15.2 &mu;g ml-1) were synthesized using the well-established Creighton method22,23 by the reduction of AgNO3 with NaBH4. AgNP polydispersity was then minimized via a 3-step TFU using a 50-nm filter (460 cm2) to remove AgNPs and AgNP-aggregates larger than 50 nm, followed by two 100-kD (200 cm2 and 20 cm2) filters to concentrate the AgNPs. Representative samples were characterized using transmission electron microscopy, UV-Vis absorption spectrophotometry, Raman spectroscopy, and inductively coupled plasma optical emission spectroscopy. The final retentate consisted of highly concentrated (4 ml, 8,539.9 &mu;g ml-1) yet lowly aggregated and homogeneous AgNPs of 1-20 nm in diameter. This corresponds to a silver concentration yield of about 62%.

Tangential Flow Ultrafiltration: A  &amp;ldquo;Green&amp;rdquo; Method for the Size Selection and Concentration of Colloidal Silver Nanoparticles

Tangential flow ultrafiltration (TFU) is a recirculation method used for the weight-based separation of biosamples. TFU was adapted to size-select (1-20 nm diameter) and highly concentrate a large volume of polydisperse silver nanoparticles (4 L of 15.2 &mu;g ml-1 down to 4 ml of 8,539.9 &mu;g ml-1) with minimal aggregation.

切向流超：A胶体银纳米粒子的大小选择与集中“绿色”方法

Nowadays, AgNPs are extensively used in the manufacture of consumer products,1 water disinfectants,2 therapeutics,1, 3 and biomedical devices4 due to ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

在对飞行性能的准确，快速测定方法的改进<em&gt;果蝇</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

An Immunofluorescent Method for Characterization of Barrett&#8217;s Esophagus Cells

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One of the primary risk factors of EAC is Barrett&#8217;s esophagus (BE), a metaplastic change of the normal squamous esophagus in response to chronic heartburn. Despite the well-established connection between EAC and BE, interrogation of the molecular events, particularly altered signaling pathways involving progression of BE to EAC, are poorly understood. Much of this is due to the lack of suitable in vitro models available to study these diseases. Recently, immortalized BE cell lines have become commercially available allowing for in vitro studies of BE. Here, we present a method for immunofluorescent staining of immortalized BE cell lines, allowing in vitro characterization of cell signaling and structure after exposure to therapeutic compounds. Application of these techniques will help develop insight into the mechanisms involved in BE to EAC progression and provide potential avenues for treatment and prevention of EAC.

An Immunofluorescent Method for Characterization of Barrett&amp;#8217;s Esophagus Cells

There is a discernible need for improved information on molecular drivers of Barrett&#8217;s Esophagus. Immunofluorescent staining is a useful technique for understanding the effects of cell signaling on cell morphology. We present a simple, effective protocol for the use of immunofluorescent staining to assess therapeutic treatment in Barrett&#8217;s Esophagus cells.

免疫荧光法Barrett食管细胞的表征

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

植物多聚核糖体剖面的简单方法

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

三维球体制作一个高效，灵活细胞聚集方法

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

ODELAY：用于酵母生长多参数定量的大规模方法

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

胞外酸性 pH 培养体系的建立

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...