Name: harvesting murine alveolar macrophages and evaluating cellular activation induced by polyanhydride nanoparticles
Uploaded: 2012-06-08T12:00:00
Description: Watch this Scientific Journal Video about Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles at JoVE.com

Forfatterne ønsker å takke den amerikanske Army Medical Research og forsyningskommando Command (Grant Numbers W81XWH-09-1-0386 og W81XWH-10-1-0806) for økonomisk støtte og Dr. Shawn Rigby fra Iowa State University flowcytometrisystemer Facility for hans ekspert teknisk assistanse.

1. Høsting alveolære makrofager (AMɸ) fra Mus hjelp Lung lavage <ol><li> Bruk egnet personlig verneutstyr (PVU) som en labfrakk, engangshansker, og riktig øyebeskyttelse. </li><li> Forbered komplett AMɸ (cAMɸ) medium før oppstart av innhøstingen. Legg 2,5 mL penicillin / streptomycin (penn / strep) løsning, 250 mL av β-mercaptoethanol (2-mercaptoethanol), og 25 ml av varme inaktivert fosteret storfe serum (FBS) til 222,25 ml Dulbelcco modifiserte Eagle Medium (DMEM). Filter-sterilisere cAMɸ medium med en 0,...

<ol>
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Polyanhydride nanopartikkel vaksine plattformer har vist effekt når det administreres intranasalt i endose regimer 5. Måling av aktivering av de fastboende phagocytic celle populasjoner i lungene forårsaket av denne vaksinen levering plattform tillater evaluering av sitt potensial evne til slutt fremme adaptive immunresponser. Nærmere bestemt, høsting alveolære makrofager fra lunge lavage fluid, og behandle dem med ulike formuleringer av nanopartikler gir innsikt i evnene ti...

<table border="1"><tbody><tr><td> Navn på reagensen </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> CAM Media </td><td></td><td></td><td></td></tr><tr><td> DMEM </td><td> Cellgro </td><td> 15-013-CV </td><td></td></tr><tr><td> 50 mm 2-mercaptoethanol </td><td> Sigma </td><td> M3148-25ML </td><td></td></tr><tr><td> Penicillin / Streptomycin 10000 mikrogram / ml </td><td> Cellgro </td><td> 30-002-CI </td><td></td></tr><tr><td> Fetal bovin serum </td><td> Atlanta Biologicals </td><td> S11150 </td><td></td></tr><tr><td> FACS Buffer </td><td></td><td></td><td></td></tr><tr><td> Natriumklorid </td><td> Fisher Scientific </td><td> S671-500 </td><td></td></tr><tr><td> Natriumfosfat </td><td> Fisher Scientific </td><td> MK7868500 </td><td></td></tr><tr><td> Kaliumklorid </td><td> Fisher Scientific </td><td> P217500 </td><td></td></tr><tr><td> Kaliumfosfat </td><td> Fisher Scientific </td><td> P288-200 </td><td></td></tr><tr><td> BSA (Bovine serum albumin) </td><td> Sigma </td><td> A7888 </td><td></td></tr><tr><td> Natriumazid </td><td> Sigma </td><td> S2002 </td><td></td></tr><tr><td> Antistoffer </td><td></td><td></td><td></td></tr><tr><td> Rat IgG </td><td> Sigma </td><td> I4341 </td><td></td></tr><tr><td> Anti-Ms CD16/32 </td><td> eBioscience </td><td> 16-0161 </td><td></td></tr><tr><td> Anti-Ms MHC II haplotype IA / IE, M5/114.15.2 klone, konjugert til fluorescein isothiocyanate (FITC) </td><td> eBioscience </td><td> 11-5321 </td><td></td></tr><tr><td> Anti-mus CD86, klon GL-1, konjugert til allophycocyanin (APC)-Cy7 </td><td> Biolegend </td><td> 105030 </td><td></td></tr><tr><td> Anti-mus CD40, klon 1C10, konjugert til APC </td><td> eBioscience </td><td> 17-0401 </td><td></td></tr><tr><td> Anti-mus CD209, klone 5H10, konjugert til Biotin </td><td> eBioscience </td><td> 13-2091 </td><td></td></tr><tr><td> Anti-mus CD11b, M1/70 klone, konjugert til Alexa Fluor 700 </td><td> eBioscience </td><td> 56-0112 </td><td></td></tr><tr><td> Anti-mus F4/80, BM8 klone, konjugert til phycoerythrin (PE)-Cy7 </td><td> eBioscience </td><td> 25-4801 </td><td></td></tr><tr><td> PE-Texas rød konjugert streptavidin </td><td> BD Biosciences </td><td> 551487 </td><td></td></tr><tr><td> Andre Rekvisita og reagenser </td><td></td><td></td><td></td></tr><tr><td> Etanol </td><td> Fisher Scientific </td><td> A405-20 </td><td> Brukes som 70% (v / v) </td></tr><tr><td> Komprimert CO 2 </td><td> Linweld </td><td> 16000060 </td><td></td></tr><tr><td> 1 ml sprøyte </td><td> BD Biosciences </td><td> 309659 </td><td></td></tr><tr><td> Sovereign 3 ½ &quot;Fr Tom Catcatheter </td><td> Kendall </td><td> 703021 </td><td></td></tr><tr><td> Biosikkerhet Cabinet </td><td> NUAIRE </td><td> Series 22 </td><td></td></tr><tr><td> Disseksjon Saks </td><td> Fisher </td><td> 138082 </td><td></td></tr><tr><td> Tang </td><td> Roboz </td><td> RS-8254 </td><td></td></tr><tr><td> PBS, 1X uten kalsium og magnesium </td><td> Cellgro </td><td> 21-040-CM </td><td></td></tr><tr><td> 15 ml Sentrifugerør med skrukork </td><td> VWR International </td><td> 21008-216 </td><td></td></tr><tr><td> Seks-brønn Tissue Culture Behandlet Plates </td><td> Costar </td><td> 3516 </td><td></td></tr><tr><td> Plastrør Racks </td><td> Nalgene </td><td> 5970 </td><td></td></tr><tr><td> Cell Scraper 24 cm </td><td> TPP </td><td> 99002 </td><td></td></tr><tr><td> 5 ml Isopor rundbunnet Tube </td><td> Falcon </td><td> 352008 </td><td></td></tr><tr><td> Pipet-Aid XL </td><td> Drummond </td><td> 4-000-105 </td><td></td></tr><tr><td> 10, 5, og 2 ml Pipettes </td><td> Fisher </td><td> 13-675 </td><td></td></tr><tr><td> 200 og 10 mL micropipettors </td><td> Gilson Pipetman </td><td> F123601 </td><td></td></tr><tr><td> 200 og 10 mL pipettespisser </td><td> Fisher </td><td> 02-707 </td><td></td></tr><tr><td> BD StabiliserendeFiksativ </td><td> BD Biosciences </td><td> 338036 </td><td></td></tr><tr><td> Isoton II Diluent </td><td> Beckman-Coulter </td><td> 8546719 </td><td></td></tr><tr><td> Zap-oglobin II lytisk reagens </td><td> Beckman-Coulter </td><td> 7546138 </td><td></td></tr><tr><td> Coulter Counter Polystyrene Rør </td><td> Beckman-Coulter </td><td> 14310-684 </td><td></td></tr><tr><td> Reagensglass </td><td> BD Biosciences </td><td> 352008 </td><td></td></tr><tr><td> Utstyr </td><td></td><td></td><td></td></tr><tr><td> Nedkjølt Sentrifuger </td><td> Labnet </td><td> 50075040 </td><td></td></tr><tr><td> Fuktet Inkubator CO 2 </td><td> Nuaire </td><td> Model Autoflyt 8500 </td><td></td></tr><tr><td> FACSCanto Flowcytometer </td><td> BD Biosciences </td><td> 338960 </td><td></td></tr><tr><td> Coulter partikkelteller Z1 </td><td> Beckman-Coulter </td><td> WS-Z1DUALPC </td><td></td></tr><tr><td> Sonicator Flytende Processing Utstyr med Microtip </td><td> Misonix </td><td> Modell nr. S-4000 </td><td></td></tr></tbody></table>

harvesting murine alveolar macrophages and evaluating cellular activation induced by polyanhydride nanoparticles

Nedbrytbare nanopartikler har dukket opp som en allsidig plattform for design og implementering av nye intranasal vaksiner mot luftveissykdommer smittsomme sykdommer. Spesielt nanopartikler polyanhydride sammensatt av alifatiske sebacic syre (SA), den aromatiske 1,6-bis (p-carboxyphenoxy) heksan (CPH), eller den amfifile 1,8-bis (p-carboxyphenoxy) -3,6-dioxaoctane (CPTEG) vise unik bulk og overflate erosjon kinetikk 1,2 og kan utnyttes til å sakte frigjøre funksjonelle biomolekyler (f.eks protein antigener, immunglobuliner etc.) in vivo 3,4,5. Disse nanopartiklene også ha egenverdi adjuvant aktivitet, noe som gjør dem til et utmerket valg for en vaksine levering plattform 6,7,8. For å belyse de mekanismer som styrer aktiveringen av medfødt immunitet etter intranasal mucosal vaksinasjon, må en vurdere de molekylære og cellulære responser av antigen presenting celler (APC) som er ansvarlige for å initiere immunreaksjoner. Dendrittiske celler er de viktigste APC funnet i gjennomføring luftveiene, mens alveolære makrofager (AMɸ) dominerer i lungene parenchyma 9,10,11. AMɸ er svært effektiv i å rydde lungene av mikrobielle patogener og celleavfall 12,13. I tillegg spiller denne celletype en verdifull rolle i transporten av mikrobielle antigener til drenering lymfeknuter, som er et viktig første skritt i initiering av en adaptiv immunrespons 9. AMɸ uttrykker også forhøyede nivåer av medfødt mønstergjenkjenning og scavenger reseptorer, skiller pro-inflammatoriske mediatorer, og prime naive T-celler 12,14. En relativt ren populasjon av AMɸ (f.eks høyere enn 80%) kan enkelt hentes via lunge lavage for studier i laboratoriet. Resident AMɸ høstet fra immun kompetente dyr gir et representativt fenotype av makrofager som vil encounter den partikkel-baserte vaksinen in vivo. Heri beskriver vi protokollene som brukes til å høste og kultur AMɸ fra mus og undersøke aktivering fenotypen av makrofager etter behandling med polyanhydride nanopartikler in vitro.

Watch this Scientific Journal Video about Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles at JoVE.com

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Heri beskriver vi protokoller for høsting murine alveolære makrofager, som er bosatt medfødte immunceller i lungene, og undersøker deres aktivisering som svar på co-kulturen med polyanhydride nanopartikler.

Høsting murint alveolære makrofager og evaluere Cellular Aktivering indusert av Polyanhydride Nanopartikler

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

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