このプロトコルは、わずか10μlの血清サンプルから、細胞外ベシクル(電気自動車)、細胞から放出された小さな膜状粒子を単離する方法を詳しく説明します。このアプローチは、超遠心分離の必要性を回避するアッセイ時間の数分しか必要とし、限られた容積のサンプルからのEVの単離を可能にする。
細胞外ベシクル(電気自動車)、細胞の様々なタイプから放出された膜状の粒子は、臨床応用のための大きな可能性を保持する。これらは、核酸およびタンパク質貨物を含む、ますます真核生物および原核生物細胞の両方によって利用される細胞間コミュニケーションの手段として認識されている。しかしながら、それらのサイズが小さいため、電気自動車の単離のための現在のプロトコルは、多くの場合、時間がかかり、面倒であり、そのような超遠心分離機のような大量の試料で高価な装置を必要とする。これらの制限に対処するために、我々は、簡単で効率的であり、そして10μlの程度の低いサンプルボリュームを必要とする電気自動車のサブグループを分離するための紙ベースの免疫親和性プラットフォームを開発した。生物学的サンプルは、化学的に特定EV表面マーカーに対して高い親和性を有する捕捉分子で変性された紙テストゾーン上に直接ピペッティングすることができる。我々は、走査電子顕微鏡(SEM)を用いてアッセイを確認、紙ベースの酵素結合immunosorbenトンアッセイ(P-ELISA)、およびトランスクリプトーム解析。これらの紙ベースのデバイスは、健康および疾患におけるEV機能の私達の理解を進める支援する診療所での電気自動車の研究と研究の設定を有効にします。
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.
細胞外ベシクルのサブグループの成功を単離するための最も重要な手順は次のとおりです。紙の1)良い選択。紙の繊維の表面上の捕捉分子の2)安定した高いカバレッジ; 3)サンプルの適切な取扱い。 4)一般的な実験室の衛生の実施を徹底する。
多孔質材料は、多くの安価機器フリーアッセイにおいて利用されている。これらは、調整可能な細孔サイズ、多目的機能、?…
The authors have nothing to disclose.
この作品は、台湾国家科学委員会grants- NSC 99から2320-B-007から005-MY2(CC)とNSC 101から2628-E-007から011-MY3(CMC)、および退役軍人将軍によって部分的にサポートされていました病院や台湾の共同研究プログラムの大学システム(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. |