Høst Muse alveolære makrofager og evaluering Cellular aktivering induceret af polyanhydridet Nanopartikler
Summary
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.
Abstract
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.) de 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'er) er ansvarlige for initiering immunresponser. Dendritiske celler er de vigtigste APC'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.
Protocol
Discussion
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Name of the reagent | Company | Catalog number | Comments |
| cAM Media | |||
| DMEM | Cellgro | 15-013-CV | |
| 50 mM 2-mercaptoethanol | Sigma | M3148-25ML | |
| Penicillin/Streptomycin 10,000 μg/ mL Solution | Cellgro | 30-002-CI | |
| Fetal Bovine Serum | Atlanta Biologicals | S11150 | |
| FACS Buffer | |||
| Sodium chloride | Fisher Scientific | S671-500 | |
| Sodium phosphate | Fisher Scientific | MK7868500 | |
| Potassium chloride | Fisher Scientific | P217500 | |
| Potassium phosphate | Fisher Scientific | P288-200 | |
| BSA (Bovine Serum Albumin) | Sigma | A7888 | |
| Sodium Azide | Sigma | S2002 | |
| Antibodies | |||
| Rat IgG | Sigma | I4341 | |
| Anti-Ms CD16/32 | eBioscience | 16-0161 | |
| Anti-Ms MHC II haplotype I-A/I-E, clone M5/114.15.2, conjugated to fluorescein isothiocyanate (FITC) | eBioscience | 11-5321 | |
| Anti-mouse CD86, clone GL-1, conjugated to allophycocyanin (APC)-Cy7 | Biolegend | 105030 | |
| Anti-mouse CD40, clone 1C10, conjugated to APC | eBioscience | 17-0401 | |
| Anti-mouse CD209, clone 5H10, conjugated to Biotin | eBioscience | 13-2091 | |
| Anti-mouse CD11b, clone M1/70, conjugated to Alexa Fluor 700 | eBioscience | 56-0112 | |
| Anti-mouse F4/80, clone BM8, conjugated to phycoerythrin (PE)-Cy7 | eBioscience | 25-4801 | |
| PE-Texas red conjugated Streptavidin | BD Biosciences | 551487 | |
| Other Supplies and Reagents | |||
| Ethanol | Fisher Scientific | A405-20 | Used as 70% (v/v) |
| Compressed CO2 | Linweld | 16000060 | |
| 1 mL Syringe | BD Biosciences | 309659 | |
| Sovereign 3 ½” Fr Tom Catcatheter | Kendall | 703021 | |
| Biosafety Cabinet | NUAIRE | Series 22 | |
| Dissection Scissors | Fisher | 138082 | |
| Forceps | Roboz | RS-8254 | |
| PBS, 1X without calcium and magnesium | Cellgro | 21-040-CM | |
| 15 mL Centrifuge Tubes with Screw Cap | VWR International | 21008-216 | |
| Six-well Tissue Culture Treated Plates | Costar | 3516 | |
| Plastic Tube Racks | Nalgene | 5970 | |
| Cell Scraper 24 cm | TPP | 99002 | |
| 5 mL Polystyrene Round-Bottom Tube | Falcon | 352008 | |
| Pipet-aid XL | Drummond | 4-000-105 | |
| 10, 5, and 2 mL Pipettes | Fisher | 13-675 | |
| 200 and 10 μL micropipettors | Gilson Pipetman | F123601 | |
| 200 and 10 μL pipette tips | Fisher | 02-707 | |
| BD Stabilizing Fixative | BD Biosciences | 338036 | |
| Isoton II Diluent | Beckman-Coulter | 8546719 | |
| Zap-oglobin II Lytic Reagent | Beckman-Coulter | 7546138 | |
| Coulter Counter Polystyrene Vials | Beckman-Coulter | 14310-684 | |
| Test Tubes | BD Biosciences | 352008 | |
| Equipment | |||
| Refrigerated Centrifuge | Labnet | 50075040 | |
| Humidified Incubator CO2 | Nuaire | Model Autoflow 8500 | |
| FACSCanto Flow Cytometer | BD Biosciences | 338960 | |
| Coulter Particle Counter Z1 | Beckman-Coulter | WS-Z1DUALPC | |
| Sonicator Liquid Processing Equipment with Microtip | Misonix | Model No. S-4000 |
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