外泌体粒径分析：Nanosight 还是ZetaView?

1. 导语

外泌体研究最近开展的如火如荼。不论是SCI文章的发表还是各类基金的申请,外泌体相关研究表现都非常亮眼。外泌体提取后,研究人员面临的一个重要问题就是,我提取的是不是外泌体?如果是的话,质量如何?

现阶段,对外泌体进行鉴定主要有三种主流的方法:电镜、粒径和Western blot分析靶标物。之前我们已经对外泌体电镜进行了总结,今天我们来看看外泌体的粒径分析。

2. 外泌体粒径分析原理

外泌体粒径分析方法为纳米颗粒跟踪分析(Nanoparticle tracking Analysis,NTA),其原理是对每个颗粒的布朗运动进行追踪和分析,结合Stockes-Einstein方程式计算出纳米颗粒的流体力学直径和浓度。NTA技术已被外泌体研究领域认可为外泌体表征手段之一。相较于其他表征方式,NTA技术的样本处理更简单、更能保证外泌体原始状态、检测速度更快。

3. 外泌体常用粒径分析仪器比较

在查阅了30多篇外泌体相关文献后,湾湾发现,目前外泌体相关文献的粒径分析主要有两种仪器,分别为Malvern’s NanoSight NS300和Particle Metrix’ ZetaView。那么这两种仪器对外泌体粒径的检测各有什么优势和劣势呢?

2019年4月,来自德国菲利普斯马尔堡大学的Elke Pogge von Strandmann课题组分别采用这两种仪器对细胞外囊泡(EV)进行NTA分析。研究人员采用透射电子显微镜(TEM)和ExoView的单粒子干涉反射传感成像系统(SP-IRIS)对人血清和细胞培养上清液的纳米球,脂质体和超速离心EV进行多次测试测量。另外,测量连续稀释和冻融循环的EV以确定每个系统的稳健性。结果发现,ZetaView在浓度检测方面测定精度更高。然而,定量TEM显示,与ZetaView相比,NanoSight NS300对EV的粒径大小检测结果更准确(%DTEM range: 79.5-134.3 vs. %DTEM range: 111.8-205.7)。两者的可重复性无显著差异。

4. SCI文章中的粒径图

4.1 10分以上的SCI文章

图1来自文献Mutant p53 cancers reprogram macrophages to tumor supporting macrophages via exosomal miR-1246. 外泌体提取方法:超速离心。粒径分析仪器:NS500。

图2来自文献Tumor-derived exosomal miR-1247-3p induces cancer-associated fibroblast activation to foster lung metastasis of liver cancer. Nature Communications. volume 9, Article number: 191 (2018). 外泌体分离方法:超速离心。粒径分析方法:Nanosight NS300.

4.2 5-10分SCI文章

图3来自文献Long non-coding RNA HOTAIR promotes exosome secretion by regulating RAB35 and SNAP23 in hepatocellular carcinoma. Mol Cancer. 2019; 18: 78. 外泌体提取方法为超速离心法;粒径分析仪器: Nanosight NS300。

图4来自文献 Hypoxic tumor-derived exosomal miR-301a mediates M2 macrophage polarization via PTEN/PI3Kr to promote pancreatic cancer metastasis. Cancer Res. 2018 Aug 15;78(16):4586-4598. 外泌体提取方法为SBI试剂盒,粒径分析仪器:Nanosight LM10

4.3 3-5分SCI文章

图5来自文献Interaction with hyaluronan matrix and miRNA cargo as contributors for in vitro potential of mesenchymal stem cell-derived extracellular vesicles in a model of human osteoarthritic synoviocytes.Stem Cell Res Ther. 2019 Mar 29;10(1):109. 本文分析的是细胞外囊泡(30-1000nm)所以EV有多个峰。EV分离方法为超速离心法。粒径分析仪器:Nanosight NS300.

图6来自文献Proteomics profiling of plasma exosomes in epithelial ovarian cancer: A potential role in the coagulation cascade, diagnosis and prognosis. Int J Oncol. 2019 May;54(5):1719-1733. 采用试剂盒exoEasy Maxi kit,粒径分析仪器:Nanosight NS300.

5. 英文方法写作

范例一

Nanoparticle tracking analysis

To analyse the size distribution of exosomes, NanoSight N300 (Malvern Instruments, Malvern, UK) was used. The samples were monitored with the use of a 640 nm laser. The exosomal sample volume was normalized to equal number (1 × 106) of XX and YY cells. The exosomal aggregations were separated using needle and syringe and simultaneously injected to the NanoSight sample cubicle. The frame rate used was 30 frames per second and NanoSight NTA3.2 software was used for data analysis.

范例二

Nanoparticle tracking analysis

The number and size of the exosomes were directly tracked by the rate of Brownian motion of exosomes using the NanoSight NS 300 system (NanoSight Technology, Malvern, UK), configured with a high-sensitivity sCMOS camera, fast video capture, and particle-tracking software (NanoSight, Amesbury, UK). The samples were diluted 150–3000 times with Dulbecco’s PBS (DPBS) without any nanoparticles to attain a concentration of 1–20 × 108 particles per milliliter for analysis. Each sample was measured in triplicate at the camera, which recorded and tracked each visible particle. Exosome numbers and size distribution were explored using the Stokes-Einstein equation.

范例三

Nanoparticle tracking analysis

Nanoparticle tracking analysis (NTA) was carried out as previously described using the NanoSight system (NanoSight; Wiltshire, UK, www.malvernpanalytical.com/en/) on EVs suspended in PBS. Vesicles were 50 fold diluted in PBS and visualized by light scattering using a conventional optical microscope aligned perpendicularly to the beam axis. NTA software tracked between frames the Brownian motion of inpidual vesicles calculating the total concentration and size through the application of Stokes-Einstein equation.

范例四

Nanoparticle tracking analysis (NTA)

Exosome concentration was analyzed using a NanoSight LM10 system (Nano sight Ltd, Navato, CA) equipped with a blue laser (405 nm). Nanoparticles were illuminated by the laser, and their movement under Brownian motion was captured for 60 seconds. The process was repeated three times. Then, all the three recorded videos were subjected to NTA using the Nanosight particle tracking software (Version NTA 3.1) to calculate exosome concentrations and size distribution.

6. 研载生物提供外泌体粒径分析服务

采用Nanosight NS300对外泌体进行粒径分析。可测量粒径范围10-1000nm(材质决定)

6.1 客户提供:

外泌体送样量为25-30μl,可以采用经过滤后的PBS进行稀释。6.2 送样要求:

1)送样时,样品须一直处于低温环境中。生物样本应避免反复冻融,新鲜外泌体样本当天或次日可到可用冰袋。冻存过的外泌体或运输时间较长,使用干冰运输。

2)样本寄送必须随样本信息服务单,服务单尽量填写完整、清晰。

6.3 服务周期:

自收到样品之日算起,3个工作日内给出测试结果。

6.4 提供结果:

1)检测报告

2)原始数据

3)粒径3D图

6.5 报告示例

注:实际检测结果受不同样本来源、样本的纯度等因素的影响,图片仅供参考。