该协议细节的方法分离细胞外囊泡(电动汽车),从细胞中释放的小的膜的颗粒,从少至10微升血清样品。这种方法绕过需要超速离心,需要测定时间仅几分钟,并且使电动车从有限体积的样品的分离。
胞外囊泡(电动汽车),从不同类型的细胞释放的膜的颗粒,持有用于临床应用的巨大潜力。它们含有核酸和蛋白质的货物和正日益被视为间通讯双方真核生物和原核生物细胞利用的手段。然而,由于它们的尺寸小,对电动汽车的隔离电流协议往往费时,繁琐,并且需要较大的样品体积和昂贵的设备,如超速离心。为了解决这些限制,我们开发了一种基于纸张免疫平台,用于分离电动汽车的亚组是容易的,高效的,并且需要的样品体积低至10微升。生物样品可以直接吸移到已经被化学修饰以捕获分子具有高亲和力的特定的EV表面标记纸测试区。我们验证该测定通过使用扫描电子显微镜(SEM),纸基酶联immunosorben吨测定(P-ELISA),和转录组分析。这些文件为基础的设备将使临床和研究设置电动车的研究,以帮助促进我们的健康和疾病EV功能的理解。
Extracellular vesicles (EVs) are heterogeneous membranous particles that range in size from 40 nm to 5,000 nm and are released actively by many cell types via different biogenesis routes1-9. They contain unique and selected subsets of DNA, RNA, proteins, and surface markers from parental cells. Their involvement in a variety of cellular processes, such as intercellular communication10, immunity modulation11, angiogenesis12, metastasis12, chemoresistance13, and the development of eye diseases9, is increasingly recognized and has spurred a great interest in their utility in diagnostic, prognostic, therapeutic, and basic biology applications.
EVs can be classically categorized as exosomes, microvesicles, apoptotic bodies, oncosomes, ectosomes, microparticles, telerosomes, prostatosomes, cardiosomes, and vexosomes, etc., based on their biogenesis or cellular origin. For example, exosomes are formed in multivesicular bodies, whereas microvesicles are generated by budding directly from plasma membrane and apoptotic vesicles are from apoptotic or necrotic cells. However, the nomenclature is still under refined, partly due to a lack of thorough understanding and characterization of EVs. Several methods have been developed to purify EVs, including ultracentrifugation14, ultrafiltration15, magnetic beads16, polymeric precipitation17-19, and microfluidic techniques20-22. The most common procedure to purify EVs involves a series of centrifugations and/or filtration to remove large debris and other cellular contaminants, followed by a final high-speed ultracentrifugation, a process that is expensive, tedious, and nonspecific14,23,24. Unfortunately, technological need for rapid and reliable isolation of EVs with satisfactory purity and efficiency is not yet met.
We have developed a paper-based immunoaffinity device that provides a simple, time- and cost-saving, yet efficient way to isolate and characterize subgroups of EVs22. Cellulose paper cut into a defined shape can be arranged and laminated using two plastic sheets with registered through-holes. In contrast to the general strategy to define the fluid boundary in paper-based devices by printing hydrophobic wax or polymers25-27, these laminated paper patterns are resistant to many organic liquids, including ethanol. Paper test zones are chemically modified to provide stable and dense coverage of capture molecules (e.g., target-specific antibodies) that have high affinity to specific surface markers on EV subgroups. Biological samples can be pipetted directly onto the paper test zones, and purified EVs are retained after rinse steps. Characterization of isolated EVs can be performed by SEM, ELISA, and transcriptomic analysis.
为胞外囊泡的子组的成功分离的最重要的步骤是:1)一个良好的纸张选择; 2)稳定和高的纸纤维的表面上的捕获分子的覆盖范围; 3)样品的妥善处理; 4)一般实验室卫生习惯。
多孔材料已经被用于许多廉价且设备无测定。它们可以具有可调孔径,多用途功能,成本低和高的表面与体积之比,允许液体的被动的芯吸。我们选择色谱纤维素纸1级主要针对其适当的孔径和低蛋白吸…
The authors have nothing to disclose.
这项工作是由台湾国科会grants- NSC 99-2320-B-007-005-MY2(CC)和NSC 101-2628-E-007-011-MY3(CMC),以及荣总部分支持医院和台湾联合研究计划大学系统(CC)。
Chromatography Paper | GE Healthcare Life Sciences | 3001-861 | Whatman® Grade 1 cellulose paper |
(3-Mercaptopropyl) trimethoxysilane | Sigma Aldrich | 175617 | This chemical reacts with water and moisture and should be applied inside a nitrogen-filled glove bag. Avoid eye and skin contact. Do not breathe fumes or inhale vapors. |
Ethanol | Fisher Scientific | BP2818 | Absolute, 200 Proof, molecular biology grade |
Bovine serum albumin (BSA) | BioShop Canada Inc. | ALB001 | Often referred to as Cohn fraction V. |
N-g-maleimidobutyryloxy succinimide ester (GMBS) | Pierce Biotechnology | 22309 | GMBS is an amine-to-sulfhydryl crosslinker. GMBS is moisture-sensitive. |
Avidin | Pierce Biotechnology | 31000 | NeutrAvidin has 4 binding sites for biotin and its pI value is 6.3, which is more neutral than native avidin |
Biotinylated mouse anti-human anti-CD63 | Ancell | 215-030 | clone AHN16.1/46-4-5 |
biotinylated annexin V | BD Biosciences | 556418 | Annxin V has a high affinity for phosphotidylserine (PS) |
Primary anti-CD9 and secondary antibody | System Biosciences | EXOAB-CD9A-1 | The secondary antibody is horseradish peroxidise-conjugated |
Serum separation tubes | BD Biosciences | 367991 | Clot activator and gel for serum separation |
Annexin V binding buffer | BD Biosciences | 556454 | 10X; dilute to 1X prior to use. |
TMB substrate reagent set | BD Biosciences | 555214 | The set contains hydrogen peroxide and 3,3’,5,5’-tetramethylbenzidine (TMB) |
RNA isolation kit | Life Technologies | AM1560 | MirVana RNA isolation kit |
Polyvinylpyrrolidone-based RNA isolation aid | Life Technologies | AM9690 | Plant RNA isolation aid contains polyvinylpyrrolidone (PVP) that binds to polysaccharides. |
RNA cleanup kit | Qiagen Inc. | 74004 | MinElute RNA cleanup kit is designed for purification of up to 45 μg RNA. |
Plasma chamber | March Instruments | PX-250 | |
Scanning electron microscope | Hitachi Ltd. | S-4300 | |
Desktop scanner | Hewlett-Packard Company | Photosmart B110 | 8-bit color images were captured. Cameras and smart phones may be also used. |
Image-record system | J&H Technology Co | GeneSys G:BOX Chemi-XX8 | 16-bit fluroscence images were captured. Fluroscence microscopes may be also used. |