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 arbeidet ble støttet av NIH/NIAID (R21AI119042) og NIH U24 eksempel sparsom analyse program (U24-AI118656).

Eksempel samlingen ble godkjent av University of Pittsburgh institusjonelle Review Board (IRB # PRO16060443, PRO10110387). Alle eksperimenter utføres under en biosafety kabinett med riktig personlig verneutstyr.
1. enheten fabrikasjon og myke Litografi
Merk: Standard myk litografi teknikker15,16 ble brukt til å opprette polydimethylsiloxane (PDMS) microchannel.
<ol><li>Bland PDMS forløperen i 10:1 forho...

<ol>
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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 treghet microfluidics lokalisere partikkel og celler et bestemt lateral sted i en mikro-kanal basert på deres størrelse5,18,19,20. På grunn av den kombinerte effekten av dekan dra kraft og treghet heis kraften i buet microchannel, store partikler eller nøytrofile (> 10 µm) er plassert inne i kanalen og små partikler, Slim aggregater og avfall mindre enn 6 µm plasseres utenfor kanalen p...

Siden celler er innkapslet i store mengder Slim i airway sekreter, har funksjonelle vurdering av leukocytter av i vitro analysen blitt hindret. Dithiothreitol (DTT) er den vanligste lyseringsbuffer distansere og homogenize sputum for fargemaskin analyse og deteksjon av meglere samtidig tålelig levedyktigheten til isolerte celler1,2. Imidlertid kan DTT forstyrre overflaten-bundet antigener i airway nøytrofile, som fører til avbrudd i nøytrofile funksjonen som elastase og myeloperoxidase (MPO) løslate2,3. Derfor har få studier av menneskelig airway nøytrofile funksjon blitt utført med perifert blod nøytrofile, som ikke kan avsløre fysiologiske kjennetegner lunge4. I mellomtiden har treghet microfluidics gjort fremskritt i isolere celler fra ulike pasienten biomatrices5,6. Likevekt mellom treghet heis styrker og Dean dra justerer partikkel/cellen avhengig av størrelse, hvilke innrømmer etikett-fri partikkel separasjon7. Vår gruppe tidligere introdusert en prøve forberedelse metode for sirkulerende tumor celler8,9, patogener i blod8, celler fra en suspensjon kultur10,11, 12, og polymorfonukleære leukocytter (PMNs) fra blodet13,14.
Her introduserer vi en protokoll for å forberede immunceller fra pasientens airway sekreter bruker lukket treghet microfluidics for en nedstrøms i vitro analysen, for eksempel nøytrofile elastase (NE) analysen. Denne metoden gir både høy konsentrasjon og utvinning, spesielt når det er en betydelig overlapping i laterale retning av cellen/partikkel som cellen/partikkel-av-begrave er fjernes, som er ofte observert i klinisk prøver. Ved resirkulering indre veggen (IW)-fokusert store partikler eller celler tilbake til input eksempel røret, partikkel eller cellen steder konsentrater i opprinnelige reservoaret, mens bakgrunn væsker med små mucin aggregat passere avfall reservoaret. Til tross for heterogene fluidic egenskaper av klinisk prøver, den foreslåtte metoden gjenoppretter konsekvent over 95% av nøytrofile fra airway sekresjon prøver som er utvannet 1000 ganger med en ren saltholdig løsning (~ 1 mL). Derimot presenterer metoden lysis et bredt utvalg av PMNs utvinningsgraden avhengig av prøven tilstand. Den foreslåtte protokollen fanger leukocytter på en etikett-fri måte med fysisk eller kjemiske avbrudd, som gir muligheten til å høste delikat celler fra klinisk utfordrende biometri med minimal invasiv prosedyrer.

<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

Airway sekreter inneholder et stort antall immun-relaterte celler, f.eks, nøytrofile, makrofager og lymfocytter, som kan brukes som en stor ressurs for å vurdere en rekke lungesykdommer, både for forskning og klinisk. Men på grunn av heterogene og tyktflytende natur pasienten Slim finnes det for øyeblikket ingen pålitelig dissosiasjon metode som ikke skade verten immunceller i pasienten airway utskillelsen. I denne forskningen introduserer vi en prøve forberedelse metode som bruker treghet microfluidics pasientens immun vurdering. Uansett heterogene fluidic egenskaper av klinisk prøvene, gjenoppretter den foreslåtte metoden mer enn 95% av nøytrofile fra airway sekresjon prøver som er utvannet 1000 ganger med ml ren saltoppløsning. Ved resirkulering konsentrert output stream til første eksempel reservoaret, tilbys det en høy konsentrasjon, gjenoppretting og renhet av immunceller; resirkulering er ansett som en avveining for enkelt kjøre sprøyte-baserte driften av treghet microfluidics. Lukket driften av spiral microfluidics gir leukocytter uten fysisk eller kjemiske forstyrrelser, som demonstrert av phorbol 12-myristate 13-acetate (PMA)-indusert elastase utgivelsen av sorterte nøytrofile.

Vi oppnådd gjennomsiktig immun-celle suspensjoner med både DTT mucolysis og microfluidics dissosiasjon (figur 3A). MicroFluidics dissosiasjon samlet 4,40 x 105 PMNs i gjennomsnitt (2,1 x 104 til 5.60 x 105 PMNs, n = 6) fra airway sekresjon prøver utvannet 1000 ganger (50 mL totalt volum) i 1 mL av ren suspensjon. Sammenlignet med den første fortynningsmiddel, 94.0% PMNs (CD66b+/CD45+) ble gjenopprette...

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 forskningen viser vi en etikett-fri nøytrofile skille metode fra klinisk airway sekreter bruker lukket drift av spiral treghet microfluidics. Den foreslåtte metoden ville utvide klinisk i vitro analyser for ulike luftveissykdommer.

Etikett-fri nøytrofile berikelse fra pasient-avledede Airway sekresjon bruker lukket treghet Microfluidics

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

Automatisert image-basert kvantifisering av nøytrofile ekstracellulære feller bruke 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

Identifikasjon av nøytrofile ekstracellulære feller i parafininnebygd feline arteriell trombi ved hjelp av immunofluorescens 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

Etikettfri kvantitativ proteomikk arbeidsflyt for oppdagelsesdrevne host-patogen interaksjoner

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

Kvantifisering av myeloperoksidase-DNA og nøytrofile elastase-DNA-komplekser fra nøytrofile ekstracellulære feller ved hjelp av en modifisert Sandwich ELISA

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