Ekstrasellüler Veziküller Yalıtım ve Karakterizasyonu için kağıt tabanlı cihazlar
Summary
Bu protokol, 10 kadar ul serum örneklerinden, hücre dışı veziküller (EVS), hücrelerden salınan küçük zar parçacıkları izole etmek için bir yöntem göstermektedir. Bu yaklaşım, ultra-santrifüj ihtiyacını önlediği deney süresi sadece birkaç dakika sürer ve sınırlı hacim örneklerinden EV'lerin izolasyonunu sağlar.
Abstract
Ekstrasellüler veziküller (AGH), hücrelerin çeşitli salınan zar parçacıklar, klinik uygulamalar için büyük bir potansiyele sahip. Onlar nükleik asit ve protein kargo içeren ve giderek ökaryotun ve prokaryot hücreler hem tarafından kullanılan arası bir iletişim aracı olarak kabul edilmektedir. Bununla birlikte, küçük boyutları için, EV'lerin izolasyonu için mevcut protokoller genellikle, zaman alıcıdır, zaman alıcıdır, ve bu, bir ultra santrifüj gibi büyük örnek hacimleri ve pahalı ekipman gerektirmektedir. Bu sınırlamaları gidermek için, biz, kolay, verimli ve 10 ul kadar düşük örnek hacimleri gerektiren EVs alt grupları ayırmak için kağıt tabanlı imünoafinite platformu geliştirdi. Biyolojik numuneler, kimyasal olarak belirli EV yüzey belirteçleri yüksek afiniteye sahip yakalama molekülü ile değiştirilmiş kağıdı test bölgeleri doğrudan pipetle edilebilir. Biz taramalı elektron mikroskobu (SEM) ile tahlil doğrulamak, kağıt-tabanlı enzim-bağlı immunosorbent tahlilleri (p-ELISA) ve transcriptome analizi. Bu kağıt-tabanlı cihazlar sağlık ve hastalık EV fonksiyonları anlayışımızı ilerletmek yardımcı klinik ve araştırma ortamında EVs çalışma sağlayacaktır.
Introduction
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.
Protocol
Representative Results
Discussion
dışı keseciklerin alt başarılı izolasyonu için en kritik adımlar şunlardır: kağıt 1) iyi bir seçim; Kağıt liflerinin yüzeyi üzerinde yakalama moleküllerinin 2) sabit ve yüksek kapsamı; 3) Numunelerin doğru kullanımı; ve 4), genel laboratuar hijyen uygulaması.
Gözenekli malzemeler, çok ucuz ve ekipman içermeyen deneylerde kullanılmıştır. Bu ayarlanabilir bir gözenek boyutu, çok yönlü özelliğe, düşük maliyet ve yüksek yüzey-hacim oranı, sıvıların p…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Bu çalışma Tayvan Ulusal Bilim Konseyi grants- MGK 99-2320-B-007-005-my2 (CC) ve MGK 101-2628-E-007-011-My3 (CMC), ve Gaziler Genel kısmen desteklenmiştir Hastaneler ve Tayvan Ortak Araştırma Programı Üniversite Sistemi (CC).
Materials
| 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. |
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