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

Gli autori desiderano ringraziare l&#39;US Army Medical Research e Materiel Command (numeri di sovvenzione W81XWH-09-1-0386 e W81XWH-10-1-0806) per il sostegno finanziario e il dottor Shawn Rigby dal Fondo Iowa State Citometria a flusso per l&#39;Università la sua assistenza tecnica di esperti.

1. Raccolta macrofagi alveolari (AMɸ) dal mouse attraverso il lavaggio del polmone <ol><li> Indossare dispositivi di protezione individuale (DPI) come un camice da laboratorio, guanti monouso e protezione per gli occhi. </li><li> Preparare completa AMɸ (cAMɸ) mezzo prima dell&#39;inizio della raccolta. Aggiungere 2,5 ml di penicillina / streptomicina (pen / strep) soluzione, 250 pl di β-mercaptoetanolo (2-mercaptoetanolo), e 25 mL di calore inattivato siero fetale bovino (FBS), a 222,25 ml di Dulbelcco Modified Ea...

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+a+Protective+Single-Dose+Intranasal+Nanoparticle-Based+Vaccine+Platform+for+Respiratory+Infectious+Diseases.">Design of a Protective Single-Dose Intranasal Nanoparticle-Based Vaccine Platform for Respiratory Infectious Diseases.</a> PLoS ONE. 6, e17642 (2011).</li><li>Petersen, L. K., Xue, L., Wannemuehler, M. J., Rajan, K., Narasimhan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+simultaneous+effect+of+polymer+chemistry+and+device+geometry+on+the+in+vitro+activation+of+murine+dendritic+cells.">The simultaneous effect of polymer chemistry and device geometry on the in vitro activation of murine dendritic cells.</a> Biomaterials. 30, 5131-5142 (2009).</li><li>Petersen, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+innate+immune+responses+in+a+pathogen-mimicking+manner+by+amphiphilic+polyanhydride+nanoparticle+adjuvants.">Activation of innate immune responses in a pathogen-mimicking manner by amphiphilic polyanhydride nanoparticle adjuvants.</a> Biomaterials. 32, 6815-6822 (2011).</li><li>Torres, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyanhydride+microparticles+enhance+dendritic+cell+antigen+presentation+and+activation.">Polyanhydride microparticles enhance dendritic cell antigen presentation and activation.</a> Acta. Biomater. 7, 2857-2864 (2011).</li><li>Kirby, A. C., Coles, M. C., Kaye, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+macrophages+transport+pathogens+to+lung+draining+lymph+nodes.">Alveolar macrophages transport pathogens to lung draining lymph nodes.</a> J. Immunol. 183, 1983-1989 (2009).</li><li>Barletta, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+compartments+in+the+mouse+lung:+Distinguishing+between+marginated,+interstitial,+and+alveolar+cells+in+response+to+injury.">Leukocyte compartments in the mouse lung: Distinguishing between marginated, interstitial, and alveolar cells in response to injury.</a> J. Immunol. Methods. , (2011).</li><li>Rubins, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+macrophages:+wielding+the+double-edged+sword+of+inflammation.">Alveolar macrophages: wielding the double-edged sword of inflammation.</a> Am. J. Respir. Crit. Care Med. 167, 103-104 (2003).</li><li>Sun, K., Gan, Y., Metzger, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Murine+Genetic+Predisposition+to+Pneumococcal+Infection+Reveals+a+Critical+Role+of+Alveolar+Macrophages+in+Maintaining+the+Sterility+of+the+Lower+Respiratory+Tract.">Analysis of Murine Genetic Predisposition to Pneumococcal Infection Reveals a Critical Role of Alveolar Macrophages in Maintaining the Sterility of the Lower Respiratory Tract.</a> Infect. Immun. 79, 1842-1847 (2011).</li><li>Morimoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+Macrophages+that+Phagocytose+Apoptotic+Neutrophils+Produce+Hepatocyte+Growth+Factor+during+Bacterial+Pneumonia+in+Mice.">Alveolar Macrophages that Phagocytose Apoptotic Neutrophils Produce Hepatocyte Growth Factor during Bacterial Pneumonia in Mice.</a> Am. J. Respir. Cell Mol. Biol. 24, 608-615 (2001).</li><li>Gordon, S., Taylor, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+and+macrophage+heterogeneity.">Monocyte and macrophage heterogeneity.</a> Nat. Rev. Immunol. 5, 953-964 (2005).</li><li>Ulery, B. 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Polianidride piattaforme vaccino nanoparticelle hanno dimostrato l&#39;efficacia se somministrato per via intranasale in regimi monodose 5. Misurazione l&#39;attivazione delle cellule fagocitiche popolazioni residenti nei polmoni indotti dalla piattaforma di mandata vaccino consente di valutare la potenziale capacità di promuovere in definitiva risposte immunitarie adattative. In particolare, la raccolta macrofagi alveolari del polmone dal liquido di lavaggio e trattarli con dive...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </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-mercaptoetanolo </td><td> Sigma </td><td> M3148-25ML </td><td></td></tr><tr><td> Penicillina / streptomicina 10.000 mg / ml di soluzione </td><td> Cellgro </td><td> 30-002-CI </td><td></td></tr><tr><td> Siero fetale bovino </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> Cloruro di sodio </td><td> Fisher Scientific </td><td> S671-500 </td><td></td></tr><tr><td> Fosfato di sodio </td><td> Fisher Scientific </td><td> MK7868500 </td><td></td></tr><tr><td> Cloruro di potassio </td><td> Fisher Scientific </td><td> P217500 </td><td></td></tr><tr><td> Fosfato di potassio </td><td> Fisher Scientific </td><td> P288-200 </td><td></td></tr><tr><td> BSA (albumina di siero bovino) </td><td> Sigma </td><td> A7888 </td><td></td></tr><tr><td> Sodio Azide </td><td> Sigma </td><td> S2002 </td><td></td></tr><tr><td> Anticorpi </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 aplotipo IA / IE, clone M5/114.15.2, coniugato con isotiocianato di fluoresceina (FITC) </td><td> EBIOScience </td><td> 11-5321 </td><td></td></tr><tr><td> Anti-mouse CD86, clone GL-1, coniugata con alloficocianina (APC)-Cy7 </td><td> Biolegend </td><td> 105030 </td><td></td></tr><tr><td> Anti-mouse CD40, clone 1C10, coniugato con APC </td><td> eBioscience </td><td> 17-0401 </td><td></td></tr><tr><td> Anti-topo CD209, clone 5H10 coniugati con biotina </td><td> eBioscience </td><td> 13-2091 </td><td></td></tr><tr><td> Anti-mouse CD11b, clone M1/70, coniugato con Alexa Fluor 700 </td><td> eBioscience </td><td> 56-0112 </td><td></td></tr><tr><td> Anti-mouse F4/80, clone BM8, coniugato con ficoeritrina (PE)-Cy7 </td><td> eBioscience </td><td> 25-4801 </td><td></td></tr><tr><td> PE-Texas red coniugato streptavidina </td><td> BD Biosciences </td><td> 551487 </td><td></td></tr><tr><td> Materiali di consumo e reagenti </td><td></td><td></td><td></td></tr><tr><td> Etanolo </td><td> Fisher Scientific </td><td> A405-20 </td><td> Utilizzato come 70% (v / v) </td></tr><tr><td> CO 2 compressa </td><td> Linweld </td><td> 16000060 </td><td></td></tr><tr><td> Siringa da 1 ml </td><td> BD Biosciences </td><td> 309659 </td><td></td></tr><tr><td> Sovereign 3 ½ &quot;Fr Catcatheter Tom </td><td> Kendall </td><td> 703021 </td><td></td></tr><tr><td> Biosicurezza Cabinet </td><td> NUAIRE </td><td> Series 22 </td><td></td></tr><tr><td> Dissezione Forbici </td><td> Pescatore </td><td> 138082 </td><td></td></tr><tr><td> Forcipe </td><td> Roboz </td><td> RS-8254 </td><td></td></tr><tr><td> PBS, 1X senza calcio e magnesio </td><td> Cellgro </td><td> 21-040-CM </td><td></td></tr><tr><td> 15 Tubi centrifuga ml con tappo a vite </td><td> VWR International </td><td> 21008-216 </td><td></td></tr><tr><td> Sei ben Cultura tessuto trattato Piatti </td><td> Costar </td><td> 3516 </td><td></td></tr><tr><td> Racks tubi in plastica </td><td> Nalgene </td><td> 5970 </td><td></td></tr><tr><td> Cella Scraper 24 centimetri </td><td> TPP </td><td> 99002 </td><td></td></tr><tr><td> 5 mL polistirene Round-Provetta a fondo </td><td> Falco </td><td> 352008 </td><td></td></tr><tr><td> Pipettare-aid XL </td><td> Drummond </td><td> 4-000-105 </td><td></td></tr><tr><td> 10, 5, e 2 ml Pipettes </td><td> Pescatore </td><td> 13-675 </td><td></td></tr><tr><td> 200 e 10 microlitri Micropipettatrici </td><td> Gilson Pipetman </td><td> F123601 </td><td></td></tr><tr><td> 200 e 10 puntali pl </td><td> Pescatore </td><td> 02-707 </td><td></td></tr><tr><td> BD di stabilizzazioneFissativo </td><td> BD Biosciences </td><td> 338036 </td><td></td></tr><tr><td> ISOTON II Diluente </td><td> Beckman-Coulter </td><td> 8546719 </td><td></td></tr><tr><td> Zap-oglobin II reagente litico </td><td> Beckman-Coulter </td><td> 7546138 </td><td></td></tr><tr><td> Fiale polistirene Coulter Counter </td><td> Beckman-Coulter </td><td> 14310-684 </td><td></td></tr><tr><td> Provette </td><td> BD Biosciences </td><td> 352008 </td><td></td></tr><tr><td> Attrezzatura </td><td></td><td></td><td></td></tr><tr><td> Centrifuga refrigerata </td><td> Labnet </td><td> 50075040 </td><td></td></tr><tr><td> Incubatore umidificato CO 2 </td><td> Nuaire </td><td> Modello Autoflow 8500 </td><td></td></tr><tr><td> FACSCanto Citofluorimetro </td><td> BD Biosciences </td><td> 338960 </td><td></td></tr><tr><td> Contatore di particelle Coulter Z1 </td><td> Beckman-Coulter </td><td> WS-Z1DUALPC </td><td></td></tr><tr><td> Sonicator Processing Equipment liquido con micropunta </td><td> Misonix </td><td> Modello No. S-4000 </td><td></td></tr></tbody></table>

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

Nanoparticelle biodegradabili sono emersi come una piattaforma versatile per la progettazione e realizzazione di nuovi vaccini intranasali contro le malattie infettive respiratorie. In particolare, polianidride nanoparticelle composto di acido alifatico sebacico (SA), il aromatico 1,6-bis (p-carbossifenossi) esano (CPH), o il anfifilo 1,8-bis (p-carbossifenossi) -3,6-dioxaoctane (CPTEG) visualizzare massa unica e cinetica di erosione superficiale e 1,2 può essere sfruttata per rilasciare lentamente biomolecole funzionali (ad esempio, antigeni proteici, immunoglobuline, ecc) in vivo 3,4,5. Queste nanoparticelle possiedono anche attività adiuvante intrinseca, che li rende una scelta eccellente per una piattaforma di distribuzione vaccino 6,7,8. Al fine di chiarire i meccanismi che regolano l&#39;attivazione dell&#39;immunità innata a seguito di vaccinazione della mucosa nasale, bisogna valutare le risposte molecolari e cellulari della p antigenecellule risentirsi (APC) responsabili per l&#39;avvio di risposte immunitarie. Le cellule dendritiche sono le principali APC si trovano nella conduzione delle vie aeree, mentre i macrofagi alveolari (AMɸ) predominano nel parenchima polmonare 9,10,11. AMɸ sono altamente efficienti in compensazione nei polmoni di agenti patogeni microbici e detriti cellulari 12,13. Inoltre, questo tipo di cellule gioca un ruolo importante nel trasporto di antigeni microbici ai linfonodi drenanti, che è un primo passo importante nella apertura di una risposta immunitaria adattativa 9. AMɸ esprimono anche livelli elevati di pattern recognition innata e recettori scavenger, secernono mediatori pro-infiammatori, e prime cellule T naive 12,14. Una popolazione relativamente pura di AMɸ (ad esempio, superiore al 80%) può essere facilmente ottenuta mediante lavaggio polmonare per studi in laboratorio. Resident AMɸ raccolte da animali immuni competenti forniscano un fenotipo rappresentante dei macrofagi che encounter la particella vaccino basato in vivo. Qui, descriviamo i protocolli utilizzati per la raccolta e la cultura AMɸ da topi ed esaminare il fenotipo attivazione dei macrofagi in seguito al trattamento con le nanoparticelle polianidride 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

Qui, descriviamo i protocolli per la raccolta murini macrofagi alveolari, che risiedono le cellule immunitarie innate nei polmoni, e di esaminare la loro attivazione in risposta a co-coltura con nanoparticelle polianidride.

Raccolta murini macrofagi alveolari e Valutare l&#39;attivazione cellulare indotta da nanoparticelle polianidride

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Bioengineering

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High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

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PLGA nanoparticelle formate da singola o doppia emulsione con Vitamina E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

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Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

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Microbiologicamente indotta Calcite Precipitazioni Mediata da<em&gt; Sporosarcina pasteurii</em

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Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

Identificazione di ossido di metallo nanoparticelle in campioni istologici da avanzato Mapping Darkfield Microscopy e iperspettrale

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Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Funzionalizzazione superficiale delle nanoparticelle di Virus di epatite E utilizzando metodi di coniugazione chimica

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Reprimere la trascrizione genica reindirizzando macchinario cellulare con modificatori epigenetici chimici

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...