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

De auteurs willen graag de US Army Medical Research en Materieel Command (Grant Numeri W81XWH-09-1-0386 en W81XWH-10-1-0806) bedanken voor financiële steun en Dr Shawn Rigby van de Iowa State University flowcytometrie faciliteit voor zijn deskundige technische bijstand.

1. Oogsten alveolaire macrofagen (AMɸ) van de muis met behulp van Lung Lavage <ol><li> Draag geschikte persoonlijke beschermingsmiddelen (PBM), zoals een laboratoriumjas, wegwerphandschoenen, en een goede oogbescherming. </li><li> Bereid volledige AMɸ (cAMɸ) medium voor de aanvang van de oogst. Voeg 2,5 ml penicilline / streptomycine (pen / strep) oplossing, 250 ul van β-mercaptoethanol (2-mercaptoethanol) en 25 ml hitte geïnactiveerd foetaal runderserum (FBS) tot 222,25 ml Dulbelcco&#39;s Gemodificeerd Eagle Med...

<ol>
	<li>Mallapragada, S. 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=Immunomodulatory+biomaterials.">Immunomodulatory biomaterials.</a> Int. J. Pharm. 364, 265-271 (2008).</li><li>Torres, M. P., Vogel, B. M., Narasimhan, B., Mallapragada, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+characterization+of+novel+polyanhydrides+with+tailored+erosion+mechanisms.">Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms.</a> J. Biomed. Mater. Res. A. 76, 102-110 (2006).</li><li>Lopac, S. K., Torres, M. P., Wilson-Welder, J. H., Wannemuehler, M. J., 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=Effect+of+polymer+chemistry+and+fabrication+method+on+protein+release+and+stability+from+polyanhydride+microspheres.">Effect of polymer chemistry and fabrication method on protein release and stability from polyanhydride microspheres.</a> Journal of Biomedical Materials Research Part B: Applied Biomaterials. 91, 938-947 (2009).</li><li>Torres, M. P., Determan, A. S., Anderson, G. L., Mallapragada, S. 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=Amphiphilic+polyanhydrides+for+protein+stabilization+and+release.">Amphiphilic polyanhydrides for protein stabilization and release.</a> Biomaterials. 28, 108-116 (2007).</li><li>Ulery, B. 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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+chemistry+influences+monocytic+uptake+of+polyanhydride+nanospheres.">Polymer chemistry influences monocytic uptake of polyanhydride nanospheres.</a> Pharm. Res. 26, 683-690 (2009).</li><li>Petersen, L. K., Sackett, C. 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=High-throughput+analysis+of+protein+stability+in+polyanhydride+nanoparticles.">High-throughput analysis of protein stability in polyanhydride nanoparticles.</a> Acta Biomater. 6, 3873-3881 (2010).</li><li>Irvin, C. G., Bates, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+lung+function+in+the+mouse:+the+challenge+of+size.">Measuring the lung function in the mouse: the challenge of size.</a> Respir. Res. 4, 4 (2003).</li></ol>

Polyanhydride nanodeeltjes vaccin platforms hebben laten zien werkzaamheid indien intranasaal toegediend in een enkele dosis regimes 5. Het meten van de activering van de bewoner fagocytaire celpopulaties in de longen geïnduceerd door het vaccin afgifte, maakt evaluatie van de potentiële mogelijkheid om uiteindelijk te bevorderen adaptieve immuunrespons. In het bijzonder, het oogsten van alveolaire macrofagen van de long lavage vloeistof en ze te behandelen met verschillende for...

<table border="1"><tbody><tr><td> Naam van het serum </td><td> Vennootschap </td><td> Catalogusnummer </td><td> Reacties </td></tr><tr><td> CAM Media </td><td></td><td></td><td></td></tr><tr><td> DMEM </td><td> Cellgro </td><td> 15 tot 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> Penicilline / streptomycine 10.000 pg / ml oplossing </td><td> Cellgro </td><td> 30-002-CI </td><td></td></tr><tr><td> Foetaal runderserum </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> Natriumchloride </td><td> Fisher Scientific </td><td> S671-500 </td><td></td></tr><tr><td> Natriumfosfaat </td><td> Fisher Scientific </td><td> MK7868500 </td><td></td></tr><tr><td> Kaliumchloride </td><td> Fisher Scientific </td><td> P217500 </td><td></td></tr><tr><td> Kaliumfosfaat </td><td> Fisher Scientific </td><td> P288-200 </td><td></td></tr><tr><td> BSA (Bovine Serum Albumine) </td><td> Sigma </td><td> A7888 </td><td></td></tr><tr><td> Natriumazide </td><td> Sigma </td><td> S2002 </td><td></td></tr><tr><td> Antilichamen </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-mevrouw CD16/32 </td><td> eBioscience </td><td> 16-0161 </td><td></td></tr><tr><td> Anti-mevrouw MHC II haplotype IA / IE, clone M5/114.15.2, geconjugeerd met fluoresceïne-isothiocyanaat (FITC) </td><td> EBIOScience </td><td> 11-5321 </td><td></td></tr><tr><td> Anti-Muis CD86, kloon GL-1, geconjugeerd aan allophycocyanin (APC)-Cy7 </td><td> Biolegend </td><td> 105030 </td><td></td></tr><tr><td> Anti-muis CD40, kloon 1C10, geconjugeerd aan APC </td><td> eBioscience </td><td> 17-0401 </td><td></td></tr><tr><td> Anti-muis CD209, een kloon 5H10, geconjugeerd aan Biotine </td><td> eBioscience </td><td> 13-2091 </td><td></td></tr><tr><td> Anti-muis CD11b, kloon M1/70, geconjugeerd met Alexa Fluor 700 </td><td> eBioscience </td><td> 56-0112 </td><td></td></tr><tr><td> Anti-muis F4/80, kloon BM8, geconjugeerd aan phycoerythrin (PE)-Cy7 </td><td> eBioscience </td><td> 25-4801 </td><td></td></tr><tr><td> PE-Texas rode geconjugeerd streptavidine </td><td> BD Biosciences </td><td> 551487 </td><td></td></tr><tr><td> Andere benodigdheden en reagentia </td><td></td><td></td><td></td></tr><tr><td> Ethanol </td><td> Fisher Scientific </td><td> A405-20 </td><td> Gebruikt als 70% (v / v) </td></tr><tr><td> Gecomprimeerde CO 2 </td><td> Linweld </td><td> 16000060 </td><td></td></tr><tr><td> 1 ml spuit </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> Bioveiligheid kabinet </td><td> NUAIRE </td><td> Series 22 </td><td></td></tr><tr><td> Dissection Schaar </td><td> Visser </td><td> 138082 </td><td></td></tr><tr><td> Pincet </td><td> Roboz </td><td> RS-8254 </td><td></td></tr><tr><td> PBS, 1X zonder calcium en magnesium </td><td> Cellgro </td><td> 21 tot 040-CM </td><td></td></tr><tr><td> 15 ml Centrifuge buizen met schroefdop </td><td> VWR International </td><td> 21008-216 </td><td></td></tr><tr><td> Zes-en Tissue Culture behandeld Platen </td><td> Costar </td><td> 3516 </td><td></td></tr><tr><td> Plastic buis Rekken </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 Polystyreen Round-benedenbuis </td><td> Valk </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 en 2 ml pipetten </td><td> Visser </td><td> 13-675 </td><td></td></tr><tr><td> 200 en 10 pi micropipettors </td><td> Gilson Pipetman </td><td> F123601 </td><td></td></tr><tr><td> 200 en 10 pi pipettips </td><td> Visser </td><td> 02-707 </td><td></td></tr><tr><td> BD stabiliserenFixatief </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 Lytische Reagens </td><td> Beckman Coulter- </td><td> 7546138 </td><td></td></tr><tr><td> Coulter Counter Polystyreen Vials </td><td> Beckman Coulter- </td><td> 14310-684 </td><td></td></tr><tr><td> Reageerbuizen </td><td> BD Biosciences </td><td> 352008 </td><td></td></tr><tr><td> Uitrusting </td><td></td><td></td><td></td></tr><tr><td> Gekoelde centrifuge </td><td> LABNET </td><td> 50075040 </td><td></td></tr><tr><td> Bevochtigde incubator 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 Particle Counter Z1 </td><td> Beckman Coulter- </td><td> WS-Z1DUALPC </td><td></td></tr><tr><td> Ultrasoonapparaat Liquid Processing Equipment met microtip </td><td> Misonix </td><td> Model No S-4000 </td><td></td></tr></tbody></table>

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

Biologisch afbreekbare nanodeeltjes naar voren zijn gekomen als een veelzijdig platform voor het ontwerp en implementatie van nieuwe intranasale vaccins tegen respiratoire infectieziekten. Specifiek, polyanhydride nanodeeltjes bestaande uit alifatische sebacinezuur (SA) de aromatische 1,6-bis (p-carboxyphenoxy) hexaan (CPH) of de amfifilische 1,8-bis (p-carboxyphenoxy) -3,6-dioxaoctane (CPTEG) weer te geven unieke bulk-en oppervlakte-erosie kinetiek 1,2 en kan worden benut om langzaam functionele biomoleculen (bijvoorbeeld eiwit antigenen, immunoglobulinen, etc.) laat in vivo 3,4,5. Deze nanodeeltjes bezitten ook intrinsieke adjuvant activiteit, waardoor ze een uitstekende keuze voor een vaccin platform voor de levering 6,7,8. Om de mechanismen die de activering van aangeboren immuniteit na intranasale mucosale vaccinatie toe te lichten, moet men de evaluatie van de moleculaire en cellulaire reacties van het antigeen phekel cellen (APC&#39;s) die verantwoordelijk is voor het initiëren van immuunreacties. Dendritische cellen zijn de voornaamste APC&#39;s gevonden in het uitvoeren van de luchtwegen, terwijl de alveolaire macrofagen (AMɸ) overheersen in het longparenchym 9,10,11. AMɸ zijn zeer efficiënt in het opruimen van de longen van microbiële ziekteverwekkers en celresten 12,13. Bovendien is dit type cel speelt een belangrijke rol in het vervoer van microbiële antigenen aan de drainerende lymfeklieren, dat is een belangrijke eerste stap in de inleiding van een adaptieve immuunrespons 9. AMɸ ook uitdrukking verhoogde niveaus van aangeboren patroonherkenning en scavenger receptoren, scheiden pro-inflammatoire mediatoren, en eerste naïeve T-cellen 12,14. Een relatief zuivere populatie van AMɸ (bijv. meer dan 80%) kan eenvoudig worden verkregen via de longen lavage voor studie in het laboratorium. Resident AMɸ geoogst van immuuncompetente dieren zorgen voor een vertegenwoordiger fenotype van de macrofagen die encounter de deeltjes gebaseerd vaccin in vivo. Hierin beschrijven we de protocollen die worden gebruikt om te oogsten en cultuur AMɸ van muizen en onderzoekt de activering fenotype van de macrofagen na behandeling met polyanhydride nanodeeltjes 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

Hierin beschrijven we protocollen voor het oogsten van muizen-alveolaire macrofagen, die aangeboren immuunsysteem cellen woonachtig zijn in de longen, en de behandeling van hun activering in reactie op de co-cultuur met polyanhydride nanodeeltjes.

Oogsten Murine alveolaire macrofagen en evalueren van cellulaire activatie geïnduceerd door polyanhydride Nanodeeltjes

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

Research

JoVE Journal

Bioengineering

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Het analyseren van cellulaire internalisatie van nanodeeltjes en bacteriën door Multi-spectrale Imaging flowcytometrie

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

High-throughput synthese van koolhydraten en functionalisering van polyanhydride Nanodeeltjes

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Evaluatie van polymere Gene Delivery Nanodeeltjes door Nanodeeltje Tracking Analyse en High-throughput Flow cytometrie

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin 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 ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Met behulp van Cell-substraat Impedantie en live cell imaging naar Real-time veranderingen in de cellulaire adhesie en De-adhesie geïnduceerd door Matrix Wijziging Meet

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

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

Microbiologisch Induced calciet Neerslag gemedieerd door<em&gt; Sporosarcina pasteurii</em

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea ...

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

Identificatie van Metal Oxide Nanodeeltjes in histologische monsters door Enhanced Darkfield Microscopie en hyperspectrale Mapping

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

Productie van Metaal Nanoparticles door Pulsed Laser Ablation in Liquids: Een Tool voor het bestuderen van de Antibacteriële Eigenschappen van Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Oppervlakte Functionalization van Hepatitis E Virus nanodeeltjes met behulp van methoden voor chemische Woordherkomst en-opbouw

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

Onderdrukken van Gene transcriptie door cellulaire machines met chemische epigenetische Modifiers omleiden

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