Denne protokollen detaljer en metode for å isolere ekstracellulære vesikler (EVS), små membran partikler frigjøres fra celler, fra så lite som 10 pl serumprøver. Denne tilnærmingen omgår behovet for ultrasentrifugering, krever bare noen få minutter av analysen tid, og muliggjør isoleringen av el-biler fra prøver av begrensede volumer.
Ekstracellulære vesikler (EVS), membran partikler frigjøres fra forskjellige typer celler, holde et stort potensial for kliniske applikasjoner. De inneholder nukleinsyre og protein last og er i økende grad anerkjent som et middel for intercellulær kommunikasjon benyttes av både eukaryote og prokaryote celler. Men på grunn av sin lille størrelse, aktuelle protokoller for isolering av elbiler er ofte tidkrevende, tungvint, og krever store prøvevolumer og dyrt utstyr, for eksempel en ultrasentrifuge. For å møte disse begrensningene, har vi utviklet et papirbasert immunoaffinitets plattform for å skille undergrupper av elbiler som er enkel, effektiv og krever prøvevolumer så lavt som 10 pl. Biologiske prøver kan pipetteres direkte på papir testsoner som er kjemisk modifisert med fangst molekyler som har høy affinitet til spesifikke markører EV overflate. Vi validere analysen ved hjelp av scanning elektronmikroskopi (SEM), papir-basert enzymbundet immunosorbent-analyser (P-ELISA), og transkriptomet analyse. Disse papirbaserte enheter vil gjøre studiet av elbiler i klinikken og forskning innstillingen til å hjelpe fremme vår forståelse av EV funksjoner i helse og sykdom.
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.
De mest kritiske trinn for vellykket isolering av undergrupper av ekstracellulære vesikler er: 1) et godt valg av papir; 2) stabil og høy dekning av fangst-molekyler på overflaten av papirfibrene; 3) riktig håndtering av prøver; og 4) generell laboratoriehygienepraksis.
Porøse materialer er blitt benyttet i mange billig og utstyr fritt assays. De kan ha avstembar porestørrelse, allsidig funksjonalitet, lav kostnad og høy overflate-til-volum-forhold tillater passiv veke av fluider. Vi…
The authors have nothing to disclose.
Dette arbeidet ble støttet delvis av Taiwan National Science Council grants- NSC 99-2320-B-007-005-MY2 (CC) og NSC 101-2628-E-007-011-My3 (CMC), og den Veterans general sykehus og University System of Taiwan Joint Research Program (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. |