Name: microfluidic platform for measuring neutrophil chemotaxis from unprocessed whole blood
Uploaded: 2014-06-03T13:00:00
Description: Watch this Scientific Journal Video about Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood at JoVE.com

Støtte fra National Institutes of Health (tilskud GM092804, DE019938) og Shriners Burns Hospital.

1.. Mikrofluid Device Fabrication <ol><li> Brug standard fotolitografisk teknikker fabrikere master mug wafer i en klasse 1000 rent rum. Mønster den første 3-um-tynde epoxy-baserede negativ fotoresistlag at definere indvandringsveje i henhold til anvisningerne fra producenten. Mønster den anden 50-um tykt lag at definere celle-loading og chemokin kamre. </li><li> Brug det mønstrede wafer at kaste polydimethylsiloxan (PDMS) enheder. Energisk blande PDMS (20 g) med initiator (2 g) i 5 min at bruge plast gaffel i sto...

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I dette arbejde har vi udviklet en mikrofluid platform til at måle neutrofil kemotaksi fra en dråbe blod (2 ul). The on-chip mekanisk filtrering af RBC&#39;er fra aktivt migrerende neutrofiler omgår behovet for besværlige celle separationsmetoder såsom massefylde gradienter 10 positiv udvælgelse 11 eller negativ selektion 12, som er tilbøjelige til at indføre artefakter ved at aktivere neutrofiler. Den mekaniske filtrering af RBC adskiller vores teknologi fra andre mikrofluid chi...

<html> Neutrofil handel spiller en afgørende rolle i fastsættelsen af fremskridt og løsning af mange inflammatoriske tilstande, herunder åreforkalkning 1, bakteriel infektion eller sepsis 2, og brænde skade 3. For deres store bidrag til sundheds-og sygdomstilstande, neutrofiltallet er del af standarden blodanalyse ofte betragtes i kliniske og forskningslaboratorier. Men på trods af at være en af de mest allestedsnærværende tests, værdien af neutrofiltallet i diagnosticering af infektion og sepsis er ofte blevet sat spørgsmålstegn 4. For eksempel er en undersøgelse af neutrofiler i brænde patienter viste, at neutrofiltallet og neutrofilmigration funktion ikke korrelerer; signalerer, at neutrofiltal alene er ikke en præcis indikator for immunstatus 3.. Selv om mere vanskelige at måle, har neutrofil funktionel kompetence blevet foreslået som mere værdifulde i en bred vifte af forhold. t &quot;&gt; vigtigt, mange af de neutrofile defekter er forbigående og ikke udløst af permanente genetiske defekter, en skelnen, der er i høj grad overset i klinikken indtil for nylig. I forbindelse med forbrændinger, kan neutrofilmigration overvåges i løbet af en patients behandling som en indikator for inflammatorisk tilstand eller infektion 3.. Traditionelle migration assays i øjeblikket anvendes i laboratoriet (Boydenkammer, Dunn kammer, mikropipette assay) ikke kan oversættes til en klinisk indstilling, fordi de kræver store mængder af blod og besværlig tidskrævende neutrofil isoleringsteknikker (tabel 1). Disse assays kan heller ikke anvendes til at overvåge kortvarige ændringer i neutrofil kemotaksi i små laboratoriedyr, såsom mus, da den mængde af blod, der er nødvendige for neutrofil isolation giver kun mulighed for en prøve og ofte endog kræver en samling af blod fra flere dyr for en enkelt assay. For eksempel kan en undersøgelse, hvori multiple vilkår og behandlinger over flere tidspunkter potentielt kunne kræve tusindvis af mus med de nuværende chemotaxis assays. Dette begrænser den biologisk grundforskning, der kan gøres for at forstå den komplekse dynamik i immun funktion i forbindelse med skade, infektion eller brænde ofte undersøgt i murine modeller 5. For at imødekomme behovet for en neutrofil funktionel analyse, der er hurtig, robust, mens der kræver minimal blodvolumen, har vi udviklet en mikrofluid enhed, der måler neutrofilkemotaksi direkte fra en lille dråbe fuldblod. Det er kendt, at mange faktorer i fuldblod, herunder serum 6 og blodplader 7, påvirker neutrofil funktion. Det er derfor hensigtsmæssigt, at helblodet microfluidic assay minimerer prøve behandling for at opretholde in vivo mikromiljø neutrofil ved måling af variationer i kemotaksi med et in vitro assay 8. Denne fremgangsmådereducerer den tid fra blodprøvetagning til neutrofilmigration analyser fra timer ved hjælp af traditionelle teknikker til få minutter (tabel 1). Helblodet microfluidic platform producerer en stabil lineær kemoattraktant gradient for længden af eksperimentet har ingen bevægelige dele og ikke kræver en ekstern trykkilde (dvs. sprøjtepumpe). Det centrale element i udformningen af ​​helblodet mikrofluid enhed er inkorporeringen af ​​røde blodlegemer (RBC) filtrering kam, der mekanisk filtrerer RBC&#39;er fra ind migration kanal af anordningen. De rigtige drejninger af denne filtrering kam forebygge behovet for størrelse udelukkelse filtrering, hvilket sandsynligvis ville blive tilstoppet af RBC og dermed blokere chemoattractant gradient fra at nå de aktivt migrerende neutrofiler i WB. Indlemmelsen af ​​fuldblod mikrofluid enhed i en 12 eller 24-brønds plade letter screening af flere mediatorer af human eller murin neutrofilkemotaksi siog traf samtidig beslutning.

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			<td height="42" style="height:42px;width:211px;">Name of Material/ Equipment</td>
			<td style="width:191px;">Company</td>
			<td style="width:179px;">Catalog Number</td>
			<td style="width:191px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:211px;">Device Fabrication</td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
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			<td height="21" style="height:21px;width:211px;">SU8&#160;</td>
			<td style="width:191px;">Microchem</td>
			<td style="width:179px;">Y131273</td>
			<td style="width:191px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Polydimethylsiloxane (PDMS)</td>
			<td style="width:191px;">Ellsworth Adhesives</td>
			<td style="width:179px;">Sylgard 184 1.1 lb. Kit</td>
			<td style="width:191px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Standard glass slides</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:179px;">125495</td>
			<td style="width:191px;">1 X 3 inches</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Glass-bottom plate</td>
			<td style="width:191px;">MatTek</td>
			<td style="width:179px;">P12G-1.5-14-F</td>
			<td style="width:191px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Harris Uni-Core, Tip Diameter 5.0mm</td>
			<td style="width:191px;">Ted Pella, Inc.</td>
			<td style="width:179px;">15081</td>
			<td style="width:191px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Harris Uni-Core, Tip Diameter 1.5 mm</td>
			<td style="width:191px;">Ted Pella, Inc.</td>
			<td style="width:179px;">15072</td>
			<td style="width:191px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Microfluidic Assay Preparation and Analysis</td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Gel-loading pipet tip</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:179px;">02-707-139</td>
			<td style="width:191px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Syringe</td>
			<td style="width:191px;">Fisher Scientfic</td>
			<td style="width:179px;">309602</td>
			<td style="width:191px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Blunt tip needle, 30g &#189; in.</td>
			<td style="width:191px;">Brico Medical Supply</td>
			<td style="width:179px;">BN3005</td>
			<td style="width:191px;"></td>
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			<td height="21" style="height:21px;width:211px;">Vacutainer, Heparin</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:211px;"></td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
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			<td height="21" style="height:21px;width:211px;">HBSS&#160;</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Human serum albumin</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:179px;">A5843-5G</td>
			<td style="width:191px;">0.2% final concentration in HBSS</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Fibronectin</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:179px;">F0895-1MG</td>
			<td style="width:191px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:211px;">fMLP</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:179px;">F3506-10MG</td>
			<td style="width:191px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:211px;">SurgiLance safety lancet, 2.2mm depth, 22 gauge</td>
			<td style="width:191px;"></td>
			<td style="width:179px;">SLN240</td>
			<td style="width:191px;"></td>
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			<td height="21" style="height:21px;width:211px;">Hoescht stain</td>
			<td style="width:191px;">Life Technologies</td>
			<td style="width:179px;">H3570</td>
			<td style="width:191px;"></td>
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			<td height="21" style="height:21px;width:211px;">Positive Control&#160;</td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:211px;">HetaSep</td>
			<td style="width:191px;">STEMCELL Technologies Inc.</td>
			<td style="width:179px;">7906</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">EasySep Human Neutrophil Enrichment Kit</td>
			<td style="width:191px;">STEMCELL Technologies Inc.</td>
			<td style="width:179px;">19257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;"></td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:211px;">Equipment</td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
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			<td height="21" style="height:21px;width:211px;">Plasma Asher</td>
			<td style="width:191px;">March Instruments</td>
			<td style="width:179px;">P-250</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Lindberg/Blue M Oven</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td style="width:179px;">13-258-30C</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Stainless Steel Precision Tweezers</td>
			<td style="width:191px;">Techni Tool</td>
			<td style="width:179px;">758TW458</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Bel-Art Scienceware Chemical-Resistant Vacuum Desiccator</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:179px;">08-594-15A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Dataplate Digital Hot Plate</td>
			<td style="width:191px;">Alpha Multiservices</td>
			<td style="width:179px;">PMC 720&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Nikon TiE inverted microscope</td>
			<td style="width:191px;">Nikon - Micro Video Instruments Inc.</td>
			<td style="width:179px;">MEA53100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">CFI Plan Fluor DL 10x na 15.2wd Objective</td>
			<td style="width:191px;">Nikon - Micro Video Instruments Inc.</td>
			<td style="width:179px;">MRH20101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Lumen 200 with 2 Meter Light Guide for Nikon</td>
			<td style="width:191px;">Nikon - Micro Video Instruments Inc.</td>
			<td style="width:179px;">500-L200NI2</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:211px;">DAPI/Hoechst/AMCA Narrow Band 32mm Exciters - 25mm Emitters</td>
			<td style="width:191px;">Chroma - Micro Video Instruments Inc.</td>
			<td style="width:179px;">31013v2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Retiga R 2000 cooled CCD Camera 1600x1200 pixels</td>
			<td style="width:191px;">Qimaging - Micro Video Instruments Inc.</td>
			<td>RET-2000R-F-M-12-C</td>
			<td></td>
		</tr>
	</tbody>
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microfluidic platform for measuring neutrophil chemotaxis from unprocessed whole blood

Neutrofiler spiller en vigtig rolle i beskyttelse mod infektioner og deres numre i blodet ofte måles i klinikken. Højere neutrofiltal i blodet er normalt en indikator for igangværende infektioner, mens lavt neutrofiltal er et advarselsskilt for højere risiko for infektioner. At udføre deres funktioner, neutrofile skal også være i stand til at bevæge sig effektivt fra blodet, hvor de tilbringer det meste af deres liv, ind i væv, hvor infektioner opstår. Følgelig kan enhver fejl i evnen af ​​neutrofiler til at migrere øge risikoen for infektioner, selv når neutrofiler er til stede i passende antal i blodet. Men måling neutrofilmigration evne i klinikken er en udfordrende opgave, som er tidskrævende, kræver stor mængde blod, og ekspertviden. For at imødegå disse begrænsninger, vi har designet en robust mikrofluid assays til neutrofilmigration, der kræver en enkelt dråbe af ubehandlet blod, omstændigvents behovet for neutrofil adskillelse og er let at kvantificere på en simpel mikroskop. I dette assay neutrofiler migrere direkte fra bloddråben, gennem små kanaler mod kilden til kemoattraktant. For at forhindre kornet strøm af røde blodlegemer gennem de samme kanaler, vi implementeret mekaniske filtre med ret vinkel vender, der selektivt blokerer fremrykningen af ​​røde blodlegemer. Vi valideret assay ved at sammenligne neutrofil migration fra bloddråber indsamlet fra stik i fingeren og veneblod. Vi sammenlignede også disse fuldblod (WB) kilder med neutrofilmigration fra prøver af oprensede neutrofiler og fundet konsekvent hastighed og retningsbestemmelse mellem de tre kilder. Denne mikrofluid platform vil gøre det muligt studiet af menneskets neutrofilmigration i klinikken og indstillingen forskning for at medvirke til at fremme vores forståelse af neutrofile funktioner i sundhed og sygdom.

Helblodet (WB) neutrofil kemotaksi assay blev valideret ved at måle ophobningen af neutrofiler i retning af en fMLP gradient (Film S1). Resultaterne bekræfter, at RBC er fanget ved filtrering kam mens neutrofiler (blå) er i stand til aktivt at migrere ud af fuldblod (figur 3A og Movie S1). Den stabile lineære kemoattraktant gradient (grøn) er dannet af fuldblod mikrofluid enhed blev bekræftet under anvendelse af FITC-mærket dextran (figur 3A inds...

Watch this Scientific Journal Video about Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood at JoVE.com

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Denne protokol beskriver en analyse designet til at måle menneskelige neutrofilkemotaksi fra en dråbe fuldblod med robust reproducerbarhed. Denne fremgangsmåde omgår behovet for neutrofil separation og kræver kun et par minutter af assay forberedelsestid. Mikrofluid chip muliggør gentagen måling af neutrofilkemotaksi over tid hos spædbørn eller små pattedyr, hvor prøvevolumen er begrænset.

Mikrofluid Platform for Måling neutrofilkemotaksi fra uforarbejdede fuldblod

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

Research

JoVE Journal

Bioengineering

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