Auf Papier basierende Vorrichtungen zur Isolierung und Charakterisierung von extrazellulärer Vesikel
Summary
Diese Protokollinformationen ein Verfahren zur extrazellulären Vesikeln (EVs), kleine membranöse Teilchen aus Zellen freigesetzt zu isolieren, von so wenig wie 10 ul Serumproben. Dieser Ansatz umgeht die Notwendigkeit von Ultrazentrifugation, nur wenige Minuten von Testzeit erfordert und ermöglicht die Isolation von Elektrofahrzeugen aus Proben von begrenzten Mengen.
Abstract
Extrazellulären Vesikeln (EVs), membranartige Partikel aus verschiedenen Arten von Zellen freigesetzt, halten ein großes Potenzial für die klinische Anwendung. Sie enthalten von Nukleinsäuren und Proteinen Ladung und werden zunehmend als Mittel zur interzellulären Kommunikation sowohl eukaryote und prokaryote Zellen verwendet erfasst. Aufgrund ihrer geringen Größe, aktuelle Protokolle zur Isolierung von Elektrofahrzeugen sind oft zeitaufwendig, umständlich und erfordert große Probenvolumina und teure Ausrüstung, wie zum Beispiel einer Ultrazentrifuge. Um diese Einschränkungen zu begegnen, haben wir eine papierbasierte Immunaffinitäts Plattform zur Trennung von Untergruppen von Elektrofahrzeugen, die einfach, effizient ist und erfordert Probenvolumen von nur 10 & mgr; l. Biologische Proben können direkt auf Papier Testzonen, die chemisch mit Fängermolekülen, die eine hohe Affinität zu bestimmten EV Oberflächenmarker haben die modifiziert wurden, pipettiert werden. Wir bestätigen die Assays unter Verwendung von Rasterelektronenmikroskopie (SEM), auf Papierbasis enzymgebundenen immunosorbent-Tests (P-ELISA) und Transkriptom-Analyse. Diese Papier-basierte Geräte ermöglichen das Studium von Elektrofahrzeugen in der Klinik und der Forschung Einstellung zu helfen unser Verständnis von EV-Funktionen in Gesundheit und Krankheit.
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
Die wichtigsten Schritte zur erfolgreichen Isolierung von Untergruppen von extrazellulären Vesikeln sind: 1) eine gute Wahl des Papiers; 2) stabile und hohe Abdeckung der Fängermoleküle auf der Oberfläche der Papierfasern; 3) die richtige Handhabung der Proben; und 4) allgemeine Laborhygienemaßnahmen.
Poröse Materialien haben sich in vielen preiswerten und Ausrüstung freien Tests genutzt. Sie haben abstimmbare Porengrße, vielseitige Funktionalität, niedrige Kosten und hohe Oberfläc…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Diese Arbeit wurde zum Teil durch die National Science Council Taiwan-Beihilfen NSC 99-2320-B-007-005-MY2 (CC) und NSC 101-2628-E-007-011-MY3 (CMC), und der Veterans General unterstützt Krankenhäuser und Universitätssystem von Taiwan Joint Research Programme (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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