Name: paper-based devices for isolation and characterization of extracellular vesicles
Uploaded: 2015-04-03T14:00:00
Description: Watch this Scientific Journal Video about Paper-based Devices for Isolation and Characterization of Extracellular Vesicles at JoVE.com

עבודה זו נתמכה בחלקו על ידי המועצה הלאומית למדע טייוואן grants- NSC 99-2320-B-007-005-MY2 (CC) והמועצה לביטחון לאומי 101-2,628-E-007-011-MY3 (CMC), ויוצאי הצבא הכללי בתי חולים ומערכת אוניברסיטת טייוואן משותף תכנית המחקר (CC).

תרשים כללי של נוהל המבצע מסופק באיור 1. שימוש בשיטות אתיות, אספנו דגימות דם מנבדקים בריאים, ואספתי דגימות הומור מימיות מחולים דרך טאיצ&#39;ונג ותיקי בית החולים הכללי (TCVGH), טאיצ&#39;ונג, טייוואן תחת IRB אושר פרוטוקולים ( IRB TCVGH מס &#39;CF11213-1). 1. י?...

<ol>
	<li>Caby, M. P., Lankar, D., Vincendeau-Scherrer, C., Raposo, G., Bonnerot, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomal-like+vesicles+are+present+in+human+blood+plasma.">Exosomal-like vesicles are present in human blood plasma.</a> Int. Immunol. 17, 879-887 (2005).</li><li>Lasser, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+saliva,+plasma+and+breast+milk+exosomes+contain+RNA:+uptake+by+macrophages.">Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages.</a> J. Transl. Med. 9, 9 (2011).</li><li>Raj, D. A. A., Fiume, I., Capasso, G., Pocsfalvi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multiplex+quantitative+proteomics+strategy+for+protein+biomarker+studies+in+urinary+exosomes.">A multiplex quantitative proteomics strategy for protein biomarker studies in urinary exosomes.</a> Kidney Int. 81, 1263-1272 (2012).</li><li>Wiggins, R. C., Glatfelter, A., Kshirsagar, B., Brukman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procoagulant+activity+in+normal+human-urine+associated+with+subcellular+particles.">Procoagulant activity in normal human-urine associated with subcellular particles.</a> Kidney Int. 29, 591-597 (1986).</li><li>Admyre, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+with+immune+modulatory+features+are+present+in+human+breast+milk.">Exosomes with immune modulatory features are present in human breast milk.</a> J. Immunol. 179, 1969-1978 (2007).</li><li>Keller, S., Ridinger, J., Rupp, A. K., Janssen, J. W. G., Altevogt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body+fluid+derived+exosomes+as+a+novel+template+for+clinical+diagnostics.">Body fluid derived exosomes as a novel template for clinical diagnostics.</a> J. Transl. Med. 9, 86 (2011).</li><li>Asea, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+protein-containing+exosomes+in+mid-trimester+amniotic+fluids.">Heat shock protein-containing exosomes in mid-trimester amniotic fluids.</a> J. Reprod. Immunol. 79, 12-17 (2008).</li><li>Bard, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+exosomes+isolated+from+human+malignant+pleural+effusions.">Proteomic analysis of exosomes isolated from human malignant pleural effusions.</a> Am. J. Resp. Cell Mol. Biol. 31, 114-121 (2004).</li><li>Perkumas, K. M., Hoffman, E. A., McKay, B. S., Allingham, R. R., Stamer, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocilin-associated+exosomes+in+human+ocular+samples.">Myocilin-associated exosomes in human ocular samples.</a> Exp. Eye Res. 84, 209-212 (2007).</li><li>Anderson, H. C., Mulhall, D., Garimella, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+extracellular+membrane+vesicles+in+the+pathogenesis+of+various+diseases,+including+cancer,+renal+diseases,+atherosclerosis,+and+arthritis.">Role of extracellular membrane vesicles in the pathogenesis of various diseases, including cancer, renal diseases, atherosclerosis, and arthritis.</a> Lab. Invest. 90, 1549-1557 (2010).</li><li>Montecalvo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+transfer+of+functional+microRNAs+between+mouse+dendritic+cells+via+exosomes.">Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes.</a> Blood. 119, 756-766 (2012).</li><li>Grange, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvesicles+released+from+human+renal+cancer+stem+cells+stimulate+angiogenesis+and+formation+of+lung+premetastatic+niche.">Microvesicles released from human renal cancer stem cells stimulate angiogenesis and formation of lung premetastatic niche.</a> Cancer Res. 71, 5346-5356 (2011).</li><li>Jaiswal, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microparticle-associated+nucleic+acids+mediate+trait+dominance+in+cancer.">Microparticle-associated nucleic acids mediate trait dominance in cancer.</a> Faseb. J. 26, 420-429 (2012).</li><li>Thery, C., Clayton, A., Amigorena, S., Raposo, G., Morgan, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current protocols in cell biology. (UNIT 3.22), (2006).</li><li>Rood, I. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+three+methods+for+isolation+of+urinary+microvesicles+to+identify+biomarkers+of+nephrotic+syndrome.">Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome.</a> Kidney Int. 78, 810-816 (2010).</li><li>Taylor, D. D., Gercel-Taylor, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+signatures+of+tumor-derived+exosomes+as+diagnostic+biomarkers+of+ovarian+cancer.">MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer.</a> Gynecol. Oncol. 110, 13-21 (2008).</li><li>Witwer, K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+sample+collection,+isolation+and+analysis+methods+in+extracellular+vesicle+research.">Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.</a> J. Extracellular Vesicles. 2, 20360 (2013).</li><li>Fernandez-Llama, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tamm-Horsfall+protein+and+urinary+exosome+isolation.">Tamm-Horsfall protein and urinary exosome isolation.</a> Kidney Int. 77, 736-742 (2010).</li><li>Alvarez, M. L., Khosroheidari, M., Ravi, R. K., DiStefano, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+protein,+microRNA,+and+mRNA+yields+using+different+methods+of+urinary+exosome+isolation+for+the+discovery+of+kidney+disease+biomarkers.">Comparison of protein, microRNA, and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers.</a> Kidney Int. 82, 1024-1032 (2012).</li><li>Chen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+isolation+and+transcriptome+analysis+of+serum+microvesicles.">Microfluidic isolation and transcriptome analysis of serum microvesicles.</a> Lab. Chip. 10, 505-511 (2010).</li><li>Davies, R. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+filtration+system+to+isolate+extracellular+vesicles+from+blood.">Microfluidic filtration system to isolate extracellular vesicles from blood.</a> Lab. Chip. 12, 5202-5210 (2012).</li><li>Chen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paper-based+immunoaffinity+devices+for+accessible+isolation+and+characterization+of+extracellular+vesicles.">Paper-based immunoaffinity devices for accessible isolation and characterization of extracellular vesicles.</a> Microfluid. Nanofluid. 16, 849-856 (2014).</li><li>Lamparski, H. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+characterization+of+clinical+grade+exosomes+derived+from+dendritic+cells.">Production and characterization of clinical grade exosomes derived from dendritic cells.</a> J. Immunol. Methods. 270, 211-226 (2002).</li><li>Cantin, R., Diou, J., Belanger, D., Tremblay, A. M., Gilbert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discrimination+between+exosomes+and+HIV-1:+Purification+of+both+vesicles+from+cell-free+supernatants.">Discrimination between exosomes and HIV-1: Purification of both vesicles from cell-free supernatants.</a> J. Immunol. Methods. 338, 21-30 (2008).</li><li>Carrilho, E., Martinez, A. W., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+wax+printing:+a+simple+micropatterning+process+for+paper-based+microfluidics.">Understanding wax printing: a simple micropatterning process for paper-based microfluidics.</a> Anal. Chem. 81, 7091-7095 (2009).</li><li>Dungchai, W., Chailapakul, O., Henry, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+detection+for+paper-based+microfluidics.">Electrochemical detection for paper-based microfluidics.</a> Anal. Chem. 81, 5821-5826 (2009).</li><li>Glavan, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Omniphobic+'R-F+paper'+produced+by+silanization+of+paper+with+fluoroalkyltrichlorosilanes.">Omniphobic 'R-F paper' produced by silanization of paper with fluoroalkyltrichlorosilanes.</a> Adv. Funct. Mater. 24, 60-70 (2014).</li><li>Usami, S., Chen, H. H., Zhao, Y. H., Chien, S., Skalak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+construction+of+a+linear+shear-stress+flow+chamber.">Design and construction of a linear shear-stress flow chamber.</a> Ann. Biomed. Eng. 21, 77-83 (1993).</li><li>Murthy, S. K., Sin, A., Tompkins, R. G., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+flow+and+surface+conditions+on+human+lymphocyte+isolation+using+microfluidic+chambers.">Effect of flow and surface conditions on human lymphocyte isolation using microfluidic chambers.</a> Langmuir. 20, 11649-11655 (2004).</li><li>Cras, J. J., Rowe-Taitt, C. A., Nivens, D. A., Ligler, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+chemical+cleaning+methods+of+glass+in+preparation+for+silanization.">Comparison of chemical cleaning methods of glass in preparation for silanization.</a> Biosens. Bioelectron. 14, 683-688 (1999).</li><li>Thery, C., Zitvogel, L., Amigorena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes:+composition,+biogenesis+and+function.">Exosomes: composition, biogenesis and function.</a> Nat. Rev. Immunol. 2, 569-579 (2002).</li><li>Scanu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulated+T+cells+generate+microparticles,+which+mimic+cellular+contact+activation+of+human+monocytes:+differential+regulation+of+pro-+and+anti-inflammatory+cytokine+production+by+high-density+lipoproteins.">Stimulated T cells generate microparticles, which mimic cellular contact activation of human monocytes: differential regulation of pro- and anti-inflammatory cytokine production by high-density lipoproteins.</a> J. Leukocyte Biol. 83, 921-927 (2008).</li><li>Inal, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvesicles+in+health+and+disease.">Microvesicles in health and disease.</a> Arch. Immunol. Ther. Ex. 60, 107-121 (2012).</li><li>Fridley, G. E., Holstein, C. A., Oza, S. B., Yager, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+nitrocellulose+as+a+material+for+bioassays.">The evolution of nitrocellulose as a material for bioassays.</a> Mrs Bull. 38, 326-330 (2013).</li><li>Gyorgy, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+vesicles,+current+state-of-the-art:+emerging+role+of+extracellular+vesicles.">Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles.</a> Cell. Mol. Life Sci. 68, 2667-2688 (2011).</li><li>Missoum, K., Belgacem, M. N., Bras, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanofibrillated+cellulose+surface+modification:+a+review.">Nanofibrillated cellulose surface modification: a review.</a> Materials. 6, 1745-1766 (2013).</li><li>Kalia, S., Boufi, S., Celli, A., Kango, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanofibrillated+cellulose:+surface+modification+and+potential+applications.">Nanofibrillated cellulose: surface modification and potential applications.</a> Colloid Polym. Sci. 292, 5-31 (2014).</li></ol>

השלבים הקריטיים ביותר לבידוד מוצלח של תת-קבוצות של שלפוחית ​​תאית הם: 1) בחירה טובה של נייר; 2) כיסוי יציב וגבוה של מולקולות ללכוד על פני השטח של סיבי נייר; טיפול נכון של דגימות 3); ו 4) תרגול היגיינה מעבדה כללית. חומרים נקבוביים כבר נ?...

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.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Chromatography Paper</td>
			<td>GE Healthcare Life Sciences</td>
			<td>3001-861</td>
			<td>&#160;Whatman&#174; Grade 1 cellulose paper</td>
		</tr>
		<tr>
			<td>(3-Mercaptopropyl) trimethoxysilane</td>
			<td>Sigma Aldrich</td>
			<td>175617</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818</td>
			<td>Absolute, 200 Proof, molecular biology grade</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>BioShop Canada Inc.</td>
			<td>ALB001</td>
			<td>Often referred to as Cohn fraction V.</td>
		</tr>
		<tr>
			<td>N-g-maleimidobutyryloxy succinimide ester (GMBS)</td>
			<td>Pierce Biotechnology</td>
			<td>22309</td>
			<td>GMBS is an amine-to-sulfhydryl crosslinker. GMBS is moisture-sensitive.</td>
		</tr>
		<tr>
			<td>Avidin</td>
			<td>Pierce Biotechnology</td>
			<td>31000</td>
			<td>NeutrAvidin has 4 binding sites for biotin and its pI value is 6.3, which is more neutral than native avidin</td>
		</tr>
		<tr>
			<td>Biotinylated mouse anti-human anti-CD63</td>
			<td>Ancell</td>
			<td>215-030</td>
			<td>clone AHN16.1/46-4-5</td>
		</tr>
		<tr>
			<td>biotinylated annexin V</td>
			<td>BD Biosciences</td>
			<td>556418</td>
			<td>Annxin V has a high affinity for phosphotidylserine (PS)</td>
		</tr>
		<tr>
			<td>Primary anti-CD9 and secondary antibody</td>
			<td>System Biosciences</td>
			<td>EXOAB-CD9A-1</td>
			<td>The secondary antibody is horseradish peroxidise-conjugated</td>
		</tr>
		<tr>
			<td>Serum separation tubes</td>
			<td>BD Biosciences</td>
			<td>367991</td>
			<td>Clot activator and gel for serum separation</td>
		</tr>
		<tr>
			<td>Annexin V binding buffer</td>
			<td>BD Biosciences</td>
			<td>556454</td>
			<td>10X; dilute to 1X prior to use.</td>
		</tr>
		<tr>
			<td>TMB substrate reagent set</td>
			<td>BD Biosciences</td>
			<td>555214</td>
			<td>The set contains hydrogen peroxide and 3,3&#8217;,5,5&#8217;-tetramethylbenzidine (TMB)</td>
		</tr>
		<tr>
			<td>RNA isolation kit</td>
			<td>Life Technologies</td>
			<td>AM1560</td>
			<td>MirVana RNA isolation kit</td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone-based RNA isolation aid</td>
			<td>Life Technologies</td>
			<td>AM9690</td>
			<td>Plant RNA isolation aid contains polyvinylpyrrolidone (PVP) that binds to polysaccharides.</td>
		</tr>
		<tr>
			<td>RNA cleanup kit</td>
			<td>Qiagen Inc.</td>
			<td>74004</td>
			<td>MinElute RNA cleanup kit is designed for purification of up to 45 &#956;g RNA.</td>
		</tr>
		<tr>
			<td>Plasma chamber</td>
			<td>March Instruments</td>
			<td>PX-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscope</td>
			<td>Hitachi Ltd.</td>
			<td>S-4300</td>
			<td></td>
		</tr>
		<tr>
			<td>Desktop scanner</td>
			<td>Hewlett-Packard Company</td>
			<td>Photosmart B110</td>
			<td>8-bit color images were captured. Cameras and smart phones may be also used.</td>
		</tr>
		<tr>
			<td>Image-record system</td>
			<td>J&#38;H Technology Co</td>
			<td>GeneSys G:BOX Chemi-XX8</td>
			<td>16-bit fluroscence images were captured. Fluroscence microscopes may be also used.</td>
		</tr>
	</tbody>
</table>

paper-based devices for isolation and characterization of extracellular vesicles

שלפוחית ​​תאית (EVS), חלקיקים קרומיים שוחררו מסוגים שונים של תאים, להחזיק פוטנציאל גדול עבור יישומים קליניים. הם מכילים מטען חומצת גרעין וחלבונים ומוכרים יותר ויותר כאמצעי לתקשורת בין תאית מנוצלת על ידי שני eukaryote ותאי פרוקריוטים. עם זאת, בשל גודלם הקטן, פרוטוקולים הנוכחיים לבידוד של כלי רכב חשמליים הם בדרך כלל זמן רב, מסורבל, ודורשים כמויות גדולות מדגם וציוד יקר, כגון ultracentrifuge. כדי לתת מענה למגבלות אלה, שפיתחנו פלטפורמת immunoaffinity מבוססת נייר להפרדה תת-קבוצות של כלי רכב חשמליים שהיא קל, יעיל, ודורש כרכי מדגם נמוכים כמו 10 μl. ניתן pipetted דגימות ביולוגיות ישירות על אזורי מבחן נייר ששונו כימי עם מולקולות ללכוד שיש לי זיקה גבוהה לסמני משטח EV ספציפיים. immunosorben צמוד אנזים אנו לאמת את assay באמצעות מיקרוסקופ אלקטרונים סורק (SEM), המבוסס על ניירמבחני t (P-ELISA), וניתוח transcriptome. מכשירים מבוססי נייר אלה יאפשר המחקר של כלי רכב חשמליים במרפאת והגדרת המחקר כדי לעזור לקדם את ההבנה של פונקציות EV בבריאות ובחוליים שלנו.

היכולת של מכשיר נייר לבודד את תת-קבוצות של כלי רכב חשמלי ביעילות מסתמכת על ההכרה שלה רגישה וספציפית של סמני משטח EV. השינוי היציב של סיבי נייר עם מולקולות ללכוד מושגת באמצעות הכימיה avidin ביוטין כפי שתואר במקומות אחרים 28-30. היעילות של נטיה הכימית ושל שיטת physisorption נב...

Watch this Scientific Journal Video about Paper-based Devices for Isolation and Characterization of Extracellular Vesicles at JoVE.com

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

פרוטוקול זה מפרט שיטה לבודד שלפוחית ​​תאית (EVS), חלקיקים קרומיים קטנים שוחררו מהתאים, מ קטן כמו 10 דגימות סרום μl. גישה זו עוקפת את הצורך בultracentrifugation, דורשת רק כמה דקות של זמן assay, ומאפשרת בידוד של כלי רכב חשמלי מדגימות של כרכים מוגבלים.

מכשירים מבוססי נייר לבידוד ואפיון של תאית שלפוחית

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain ...

Research

JoVE Journal

Bioengineering

Manufacturing Devices and Instruments for Easier Rat Liver Transplantation

התקני ייצור מכשירים להשתלת כבד חולדה קלה יותר

Orthotopic rat liver transplantation is a popular model, which has been shown in a recent JoVE paper with the use of the "quick-linker" device. This ...

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

כימות וגודל-פרופיל של תאית שלפוחית ​​באמצעות Pulse חישה מתכוונן התנגדותי

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

ייצור של מכשירי microfluidic נייר מבוססים תלת ממדי עבור Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

זיהוי של אנדוטוקסין ניסוחים-Nano באמצעות מבחני Lysate (LAL) Amoebocyte Limulus

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Evaluation of the Storage Stability of Extracellular Vesicles

הערכת יציבות האחסון של שלפוחיות ומסחטות

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

התקני שדה חשמליים ומגנטיים לגירוי רקמות ביולוגיות

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

תיוג של שלל חוץ-תאי לניטור הגירה וספיגה של סחוס

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their ...

Improving Reproducibility to Meet Minimal Information for Studies of Extracellular Vesicles 2018 Guidelines in Nanoparticle Tracking Analysis

שיפור רבייה כדי לענות על מידע מינימלי למחקרים של שלפוחיות חוץ-תאיות 2018 הנחיות בניתוח מעקב חלקיקים

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many ...

Design to Implementation Study for Development and Patient Validation of Paper-Based Toehold Switch Diagnostics

תכנון ליישום מחקר לפיתוח ותיקוף מטופלים של אבחון מתגי Toehold מבוסס נייר

Access to low-burden molecular diagnostics that can be deployed into the community for testing is increasingly important and has meaningful wider ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

הערכה של טיפול אפנון התכווצות הלב בקרדיומיוציטים דו-ממדיים שמקורם בתאי גזע אנושיים

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...