Name: label-free neutrophil enrichment from patient-derived airway secretion using closed-loop inertial microfluidics
Uploaded: 2018-06-07T14:30:00
Description: Watch this Scientific Journal Video about Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics at JoVE.com

Dette arbejde blev støttet af NIH/NIAID (R21AI119042) samt NIH U24 prøve besparende assay program (U24-AI118656).

Samlingen prøven blev godkendt af University of Pittsburgh institutionelle Review Board (IRB # PRO16060443, PRO10110387). Alle forsøg udføres under en biosikkerhed kabinet med de korrekte personlige værnemidler.
1. enhed fabrikation og bløde litografi
Bemærk: Standard bløde litografi teknikker15,16 blev brugt til at oprette Polydimethylsiloxan (PDMS) microchannel.
<ol><li>Bland PDMS forløber i forh...

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dithiothreitol+has+a+dose-response+effect+on+cell+surface+antigen+expression.">Dithiothreitol has a dose-response effect on cell surface antigen expression.</a> J Allergy Clin Immunol. 103 (5 Pt 1), 873-876 (1999).</li><li>Usher, L. 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T., Han, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+detection+and+drug-resistance+profiling+of+bacteremias+using+inertial+microfluidics.">Direct detection and drug-resistance profiling of bacteremias using inertial microfluidics.</a> Lab Chip. 15 (10), 2297-2307 (2015).</li><li>Warkiani, M. E., 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=Ultra-fast,+label-free+isolation+of+circulating+tumor+cells+from+blood+using+spiral+microfluidics.">Ultra-fast, label-free isolation of circulating tumor cells from blood using spiral microfluidics.</a> Nat Protoc. 11 (1), 134-148 (2016).</li><li>Warkiani, M. E., Tay, A. 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A., Han, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+leukocytes+from+blood+using+spiral+channel+with+trapezoid+cross-section.">Separation of leukocytes from blood using spiral channel with trapezoid cross-section.</a> Anal Chem. 84 (21), 9324-9331 (2012).</li><li>Guan, 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=Spiral+microchannel+with+rectangular+and+trapezoidal+cross-sections+for+size+based+particle+separation.">Spiral microchannel with rectangular and trapezoidal cross-sections for size based particle separation.</a> Sci Rep. 3, 1475 (2013).</li><li>Kotz, K., Cheng, X., 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=PDMS+Device+Fabrication+and+Surface+Modification.">PDMS Device Fabrication and Surface Modification.</a> J Vis Exp. (8), e319 (2007).</li><li>Duffy, D. C., McDonald, J. C., Schueller, O. J. A., 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=Rapid+Prototyping+of+Microfluidic+Systems+in+Poly(dimethylsiloxane).">Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane).</a> Analytical Chemistry. 70 (23), 4974-4984 (1998).</li><li>Ryu, H., 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=Patient-Derived+Airway+Secretion+Dissociation+Technique+To+Isolate+and+Concentrate+Immune+Cells+Using+Closed-Loop+Inertial+Microfluidics.">Patient-Derived Airway Secretion Dissociation Technique To Isolate and Concentrate Immune Cells Using Closed-Loop Inertial Microfluidics.</a> Anal Chem. 89 (10), 5549-5556 (2017).</li><li>Mach, A. J., Di Carlo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+scalable+blood+filtration+device+using+inertial+microfluidics.">Continuous scalable blood filtration device using inertial microfluidics.</a> Biotechnol Bioeng. 107 (2), 302-311 (2010).</li><li>Di Carlo, D., Irimia, D., Tompkins, R. 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IX. Elastinolytic activity in granules of human polymorphonuclear leukocytes.</a> J Exp Med. 128 (5), 1137-1155 (1968).</li><li>Kawabata, K., Hagio, T., Matsuoka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+neutrophil+elastase+in+acute+lung+injury.">The role of neutrophil elastase in acute lung injury.</a> Eur J Pharmacol. 451 (1), 1-10 (2002).</li><li>Rubin, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastic+Bronchitis.">Plastic Bronchitis.</a> Clin Chest Med. 37 (3), 405-408 (2016).</li><li>Kokot, K., Teschner, M., Schaefer, R. 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I inertial mikrofluidik lokalisere partikel og celler på en bestemt lateral position i en mikro-kanal er baseret på deres størrelse5,18,19,20. På grund af den kombinerede effekt af dekanen træk kraft og inertial lift kraften i buede microchannel, store partikler eller neutrofile (> 10 µm) er placeret inde i kanal og små partikler, Slim aggregater og snavs mindre end 6 µm er placeret ude...

Da cellerne er indkapslet i en stor mængde slim i luftvejene sekreter, er funktionel vurdering af leukocytter ved en in vitro- assay blevet forhindret. Dithiothreitol (DTT) er den mest almindelige lysisbuffer at adskille og homogeniseres sputum cytologiske analyse og påvisning af mæglere samtidig tolerable levedygtighed af isolerede celler1,2. Dog kan DTT interferere med overflade-bundet antigener af luftvejene neutrofiler, hvilket resulterer i afbrydelse af neutrofile funktion såsom elastase og myeloperoxidase (MPO) frigive2,3. Derfor er få undersøgelser af menneskers luftveje neutrofile funktion blevet gennemført med perifert blod neutrofiler, som ikke kan afsløre de fysiologiske egenskaber af pulmonal4. I mellemtiden, inertial mikrofluidik har gjort fremskridt i isolere celler fra forskellige patient biomatrices5,6. Ligevægt mellem inertial lift styrker og Dean træk justerer partikel/celle efter deres størrelse, som giver mulighed for etiket-fri partikel adskillelse7. Vores gruppe tidligere indført præparationsmetode prøve for cirkulerende tumor celler8,9, patogener i blod8, celler fra en suspension kultur10,11, 12, og polymorfnukleære leukocytter (PMNs) fra blod13,14.
Her introducerer vi en protokol for at forberede immun celler fra patientens luftveje sekreter med lukket kredsløb inertial mikrofluidik for en downstream i vitro assay, såsom neutrofile elastase (NE) assay. Denne metode giver både høj koncentration og nyttiggørelse, især når der findes en betydelig overlapning i lateral retning af den celle/partikel hvorfra den celle/partikel-af-interesse er at blive fjernet, der er almindeligt observeret i kliniske prøver. Af recirkulerende indre væg (IW)-fokuseret store partikler eller celler tilbage til input prøveglas, partikel eller celle-af-interesse koncentrerer sig i den oprindelige reservoir, mens baggrunden væsker med små mucin aggregater passere gennem affald reservoiret. Trods de heterogene fluidic egenskaber af kliniske prøver, den foreslåede metode genopretter konsekvent over 95% af neutrofiler fra luftvejene sekretion prøver, der er fortyndet 1.000-fold med en ren saltvandsopløsning (~ 1 mL). Derimod præsenterer metoden lysis en bred vifte PMNs genfindingsprocent afhængigt af prøven tilstand. Den foreslåede protokol indfanger leukocytter i en etiket-fri måde med ingen fysiske eller kemiske forstyrrelser, som giver mulighed for at høste sarte celler fra klinisk udfordrende biometri med minimalt invasive procedurer.

<table>
	<tbody>
		<tr>
			<td>PDMS precursor</td>
			<td>Dow corning</td>
			<td>184 SIL ELAST KIT 3.9KG</td>
			<td>10:1 ratio of base and curing agent</td>
		</tr>
		<tr>
			<td>VWR gravity convection oven</td>
			<td>VWR</td>
			<td>414005-128</td>
			<td>PDMS precursor to be cured in 90 deg.</td>
		</tr>
		<tr>
			<td>100mm petri dish</td>
			<td>VWR</td>
			<td>89000-324</td>
			<td>Fabrication of PDMS Supporting layer</td>
		</tr>
		<tr>
			<td>Harris Uni-core puncher</td>
			<td>Sigma-aldrich</td>
			<td>WHAWB100076</td>
			<td>2mm diameter or other depending on the tubing size</td>
		</tr>
		<tr>
			<td>Air plasma machine</td>
			<td>Femto Science</td>
			<td>Cute</td>
			<td>Surface plasma treatment for PDMS device to bottom base.</td>
		</tr>
		<tr>
			<td>2&#8221; x 3&#8221; glass slide</td>
			<td>TED PELLA, INC.</td>
			<td>2195</td>
			<td>To support PDMS device</td>
		</tr>
		<tr>
			<td>Masterflex spooled platinum-cured silicone tubing, L/S 14</td>
			<td>Cole-Parmer</td>
			<td>EW-96410-14</td>
			<td>Tubing for microfluidics and peristlatic pump</td>
		</tr>
		<tr>
			<td>1/16 inch Luer connector, male</td>
			<td>Harvard apparatus</td>
			<td>PC2 72-1443</td>
			<td>Connector for fluid guide</td>
		</tr>
		<tr>
			<td>50mL Falcon tube</td>
			<td>Corning</td>
			<td>21008-940</td>
			<td>sample collection &#38; preparation</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline, 1X Without Calcium and Magnesium</td>
			<td>Corning</td>
			<td>45000-446&#160;</td>
			<td>Buffer solution to dilute sample</td>
		</tr>
		<tr>
			<td>Halyard Closed suction Catheter, Elbow, 14F/ channel 4.67mm</td>
			<td>HALYARD HEALTH</td>
			<td>22113</td>
			<td>Tracheal seceation suction catheter</td>
		</tr>
		<tr>
			<td>0.9% Sterile Normal saline, 10mL pre-filled syringe</td>
			<td>BD PosiFlush</td>
			<td>NHRIC: 8290-306547</td>
			<td>For tracheal seceation collection from the patients</td>
		</tr>
		<tr>
			<td>SecurTainer&#8482; III Specimen Containers, 20mL</td>
			<td>Simport</td>
			<td>1176R36</td>
			<td>Sterile sputum (airway secretion) collection container</td>
		</tr>
		<tr>
			<td>Syringe with Luer-Lok Tip, 10mL</td>
			<td>BD</td>
			<td>BD309604</td>
			<td>To pipette homogenize the mucus sample and reach the bottom of sample tube</td>
		</tr>
		<tr>
			<td>BD&#160; Blunt Fill Needle, with BD Luer-Lok&#160; Tip</td>
			<td>BD</td>
			<td></td>
			<td>To pipette homogenize the mucus sample and reach the bottom of sample tube</td>
		</tr>
		<tr>
			<td>40&#181;m nylon cell strainer&#160;</td>
			<td>Falcon</td>
			<td>21008-949</td>
			<td>To remove large chunk or blood clots, which can block the microfluidics access hole or the channel.</td>
		</tr>
		<tr>
			<td>Peristaltic pump (Masterflex L/S Digital Drive)</td>
			<td>Cole-Parmer</td>
			<td>HV-07522-30</td>
			<td>operation of microfluidics</td>
		</tr>
		<tr>
			<td>BD LSR II flow cytometer</td>
			<td>BD Bioscience</td>
			<td>LSR II flow cytometer</td>
			<td>Quantification of cell recovery ratio</td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD66b monoclonal antibody</td>
			<td>BD Bioscience</td>
			<td>561927</td>
			<td>Immunostaining of neutrophils for Flow cytometer analysis</td>
		</tr>
		<tr>
			<td>Allophycocyanin (APC)-conjugated mouse anti-human CD45 monoclonal antibody</td>
			<td>BD Bioscience</td>
			<td>561864</td>
			<td>Immunostaining of neutrophils for Flow cytometer analysis</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>Thermo Fisher scientific</td>
			<td>Varioskan</td>
			<td>Plate reader for neutrophil elastase assay, ex485/em525</td>
		</tr>
		<tr>
			<td>Neutrophil elastase assay kit</td>
			<td>Cayman Chemical</td>
			<td>600610</td>
			<td>Neutrophil functionality assessment</td>
		</tr>
		<tr>
			<td>Fluoresbrite YG Microspheres 10.0&#181;m</td>
			<td>PolyScience, Inc.</td>
			<td>18140-2</td>
			<td>Fluorescent particles to express white blood cell trajectory in microfluidics</td>
		</tr>
	</tbody>
</table>

label-free neutrophil enrichment from patient-derived airway secretion using closed-loop inertial microfluidics

Luftveje sekreter indeholder et stort antal immun-relaterede celler, fx, neutrofiler, makrofager og lymfocytter, som kan bruges som en stor ressource til at evaluere en række forskellige lungesygdomme, både til forskning og kliniske formål. Men på grund af de heterogene og tyktflydende karakter af patient slim, der er i øjeblikket ingen pålidelig dissociation metode, der ikke skader vært immunceller i patientens luftveje sekretion. I denne forskning introducere vi præparationsmetode stikprøve, der bruger inertial mikrofluidik for patientens immunforsvar vurdering. Uanset de heterogene fluidic egenskaber af de kliniske prøver, den foreslåede metode genindvinder mere end 95% af neutrofiler fra luftvejene sekretion prøver, der er fortyndet 1.000-fold med milliliter af rent saltvand. Af recirkulerende koncentreret outputstream til den oprindelige stikprøve reservoir, leveres en høj koncentration, recovery og renhed af immunceller; recirkulation anses et trade-off for single-run sprøjte-baseret funktion inertial mikrofluidik. Lukket kredsløb driften af spiral mikrofluidik giver leukocytter uden fysiske eller kemiske forstyrrelser, som det fremgår af phorbol 12-myristate 13-acetat (PMA)-induceret elastase frigivelse af sorterede neutrofiler.

Vi opnåede gennemsigtig immun-celle suspensioner med begge DTT mucolysis og mikrofluidik dissociation (figur 3A). Mikrofluidik dissociation indsamlet 4.40 x 105 PMNs i gennemsnit (2.1 x 104 til 5,60 x 105 PMNs, n = 6) fra luftvejene sekretion prøverne fortyndes 1.000-fold (50 mL samlede volumen) i 1 mL ren suspension. I forhold til den oprindelige fortyndingsmiddel, 94.0% PMNs (CD66b+/CD45+) blev genfun...

Watch this Scientific Journal Video about Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics at JoVE.com

Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics

I denne forskning viser vi en etiket-fri neutrofile adskillelse metode fra kliniske luftveje sekreter med lukket kredsløb drift af spiral inertial mikrofluidik. Den foreslåede metode ville udvide klinisk in vitro- assays til forskellige luftvejssygdomme.

Label-fri neutrofile berigelse fra Patient-afledte luftvejene sekretion ved hjælp af lukkede kredsløb Inertial mikrofluidik

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource ...

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Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Automatiseret billedbaseret kvantificering af neutrofile ekstracellulære fælder med NETQUANT

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. ...

Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy

Identifikation af neutrofile ekstracellulære fælder i paraffin-embedded feline arteriel trombose ved hjælp af Immunofluorescence Mikroskopi

Neutrophil extracellular traps (NETs), composed of cell-free DNA (cfDNA) and proteins like histones and neutrophil elastase (NE), are released by ...

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

Label-Free Kvantitative Proteomics Workflow for Discovery-Driven Host-Patogen Interaktioner

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s ...

Quantifying Myeloperoxidase-DNA and Neutrophil Elastase-DNA Complexes from Neutrophil Extracellular Traps by Using a Modified Sandwich ELISA

Kvantificering af myeloperoxidase-DNA og neutrofile elastase-DNA-komplekser fra neutrofile ekstracellulære fælder ved hjælp af en modificeret sandwich-ELISA

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures ...