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 vil gerne takke den amerikanske hær Medical Research og Materiel Kommando (Grant tal W81XWH-09-1-0386 og W81XWH-10-1-0806) for finansiel støtte og Dr. Shawn Rigby fra Iowa State University Flowcytometri facilitet for hans ekspert teknisk bistand.

1. Høst alveolære makrofager (AMɸ) fra mus ved hjælp af lungeudskylning <ol><li> Bær passende personlige værnemidler (PPE), såsom en laboratoriekittel, engangshandsker og passende øjenbeskyttelse. </li><li> Forbered komplet AMɸ (cAMɸ) medium inden påbegyndelse af høst. Tilsættes 2,5 ml penicillin / streptomycin (pen / strep) opløsning, 250 pi β-mercaptoethanol (2-mercaptoethanol), og 25 ml varmeinaktiveret føtalt bovint serum (FBS) til 222,25 ml Dulbelcco s Modified Eagle Medium (DMEM). Filter-sterilis...

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Polyanhydridet nanopartikel vaccine platforme har vist effekt, når det gives intranasalt i enkeltdoser regimer 5. Måling af aktivering af de residente fagocytiske cellepopulationer i lungerne som følge af denne vaccine leveringsplatform tillader evaluering af potentielle evne til sidst fremme adaptive immunrespons. Specifikt høst alveolære makrofager fra lungeudskylning fluid og behandle dem med forskellige formuleringer af nanopartiklerne giver indsigt i de evner forskellige...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nr. </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 til 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 10.000 ug / ml opløsning </td><td> Cellgro </td><td> 30-002-CI </td><td></td></tr><tr><td> Føtalt bovint 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> Natriumchlorid </td><td> Fisher Scientific </td><td> S671-500 </td><td></td></tr><tr><td> Natriumphosphat </td><td> Fisher Scientific </td><td> MK7868500 </td><td></td></tr><tr><td> Kaliumchlorid </td><td> Fisher Scientific </td><td> P217500 </td><td></td></tr><tr><td> Kaliumphosphat </td><td> Fisher Scientific </td><td> P288-200 </td><td></td></tr><tr><td> BSA (bovint serumalbumin) </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> Rotte 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, klon M5/114.15.2, konjugeret til fluoresceinisothiocyanat (FITC) </td><td> EBIOSREnescience </td><td> 11-5321 </td><td></td></tr><tr><td> Anti-muse CD86, klon GL-1, konjugeret til allophycocyanin (APC)-Cy7 </td><td> Biolegend </td><td> 105030 </td><td></td></tr><tr><td> Anti-muse CD40, klon 1C10, konjugeret til APC </td><td> eBioscience </td><td> 17-0401 </td><td></td></tr><tr><td> Anti-muse-CD209, klon 5H10 og konjugeret til biotin </td><td> eBioscience </td><td> 13-2091 </td><td></td></tr><tr><td> Anti-mus CD11b, klon M1/70, konjugeret til Alexa Fluor 700 </td><td> eBioscience </td><td> 56-0112 </td><td></td></tr><tr><td> Anti-muse F4/80, klon BM8, konjugeret til phycoerythrin (PE)-Cy7 </td><td> eBioscience </td><td> 25-4801 </td><td></td></tr><tr><td> PE-Texas Red-konjugeret streptavidin </td><td> BD Biosciences </td><td> 551487 </td><td></td></tr><tr><td> Andre forbrugsvarer og reagenser </td><td></td><td></td><td></td></tr><tr><td> Ethanol </td><td> Fisher Scientific </td><td> A405-20 </td><td> Anvendes som 70% (v / v) </td></tr><tr><td> Komprimeret CO 2 </td><td> Linweld </td><td> 16000060 </td><td></td></tr><tr><td> 1 ml sprøjten </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> Biosikkerhed Cabinet </td><td> NUAIRE </td><td> Serie 22 </td><td></td></tr><tr><td> Dissektion 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 uden calcium og magnesium </td><td> Cellgro </td><td> 21-040-CM </td><td></td></tr><tr><td> 15 ml centrifugerør med skruelåg </td><td> VWR International </td><td> 21008-216 </td><td></td></tr><tr><td> Seks brønde Tissue Culture behandlede plader </td><td> Costar </td><td> 3516 </td><td></td></tr><tr><td> Plasticrør racks </td><td> Nalgene </td><td> 5970 </td><td></td></tr><tr><td> Celleskraber 24 cm </td><td> TPP </td><td> 99002 </td><td></td></tr><tr><td> 5 ml Polystyren rundbundet Tube </td><td> Falcon </td><td> 352008 </td><td></td></tr><tr><td> Afpipettér-støtte XL </td><td> Drummond </td><td> 4-000-105 </td><td></td></tr><tr><td> 10, 5 og 2 ml pipetter </td><td> Fisher </td><td> 13-675 </td><td></td></tr><tr><td> 200 og 10 gl micropipettors </td><td> Gilson Pipetman </td><td> F123601 </td><td></td></tr><tr><td> 200 og 10 gl pipettespidser </td><td> Fisher </td><td> 02-707 </td><td></td></tr><tr><td> BD StabiliserendeFixativ </td><td> BD Biosciences </td><td> 338036 </td><td></td></tr><tr><td> Isoton II fortyndingsmiddel </td><td> Beckman-Coulter </td><td> 8546719 </td><td></td></tr><tr><td> ZAP-oglobin II lytiske reagens </td><td> Beckman-Coulter </td><td> 7546138 </td><td></td></tr><tr><td> Coulter Counter Polystyren Hætteglas </td><td> Beckman-Coulter </td><td> 14310-684 </td><td></td></tr><tr><td> Reagensglas </td><td> BD Biosciences </td><td> 352008 </td><td></td></tr><tr><td> Udstyr </td><td></td><td></td><td></td></tr><tr><td> Kølecentrifuge </td><td> LABNET </td><td> 50075040 </td><td></td></tr><tr><td> Befugtet inkubator CO 2 </td><td> Nuaire </td><td> Model Autoflow 8500 </td><td></td></tr><tr><td> FACSCanto Flow Cytometer </td><td> BD Biosciences </td><td> 338960 </td><td></td></tr><tr><td> Coulter partikeltæller Z1 </td><td> Beckman-Coulter </td><td> WS-Z1DUALPC </td><td></td></tr><tr><td> Sonicator Liquid Processing Udstyr med mikrotip </td><td> Misonix </td><td> Model nr. S-4000 </td><td></td></tr></tbody></table>

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

Bionedbrydelige nanopartikler er opstået som en alsidig platform for design og implementering af nye intranasale vacciner mod respiratoriske smitsomme sygdomme. Specifikt polyanhydrid nanopartikler bestående af alifatiske sebacinsyre (SA), den aromatiske 1,6-bis (p-carboxyphenoxy) hexan (CPH) eller amfifile 1,8-bis (p-carboxyphenoxy) -3,6-dioxaoctan (CPTEG) udviser enestående bulk overflade nedbrydningskinetik 1,2 og kan udnyttes til langsomt at frigive funktionelle biomolekyler (f.eks proteinantigener, immunoglobuliner, etc.) in vivo 3,4,5. Disse nanopartikler har også iboende adjuvansaktivitet, hvilket gør dem til et glimrende valg for en vaccine leveringsplatform 6,7,8. For at belyse de mekanismer, der styrer aktiveringen af ​​medfødte immunitet efter intranasal slimhinde vaccination, skal man vurdere de molekylære og cellulære reaktioner antigenet presenting celler (APC&#39;er) er ansvarlige for initiering immunresponser. Dendritiske celler er de vigtigste APC&#39;erne findes i ledende luftveje, mens alveolære makrofager (AMɸ) dominerer i lungeparenkymet 9,10,11. AMɸ er meget effektive til at rense lungerne af mikrobielle patogener og celledebris 12,13. Hertil kommer, spiller denne celletype en værdifuld rolle i forbindelse med transport af mikrobielle antigener til de drænende lymfeknuder, hvilket er et vigtigt første skridt i iværksættelsen af et adaptivt immunrespons 9. AMɸ udtrykker også forhøjede niveauer af medfødte mønstergenkendelse og scavenger-receptorer, udskiller pro-inflammatoriske mediatorer, og prime naive T-celler 12,14. En relativt ren population af AMɸ (fx større end 80%) kan let opnås via lungeudskylning til undersøgelse i laboratoriet. Hjemmehørende AMɸ høstet fra immunkompetente dyr giver en repræsentativ fænotype af de makrofager, der encounter partikel-baseret vaccine in vivo. Heri, beskriver vi de protokoller, der anvendes til høst og kulturen AMɸ fra mus og undersøge aktivering fænotypen af makrofager efter behandling med polyanhydrid 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 til høstning murine alveolære makrofager, som er beliggende medfødte immunsystem-celler i lungen, og undersøge deres aktivering som reaktion på samdyrkning med polyanhydrid nanopartikler.

Høst Muse alveolære makrofager og evaluering Cellular aktivering induceret af polyanhydridet Nanopartikler

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

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