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

The authors would like to thank the U.S. Army Medical Research and Materiel Command (Grant Numbers W81XWH-09-1-0386 and W81XWH-10-1-0806) for financial support and Dr. Shawn Rigby from the Iowa State University Flow Cytometry Facility for his expert technical assistance.

1. Harvesting Alveolar Macrophages (AM&#632;) from the Mouse using Lung Lavage
<ol>
 <li>Wear appropriate personal protective equipment (PPE) such as a lab coat, disposable gloves, and proper eye protection.</li>
 <li>Prepare complete AM&#632; (cAM&#632;) medium before initiation of harvest. Add 2.5 mL of penicillin/streptomycin (pen/strep) solution, 250 &mu;L of &beta;-mercaptoethanol (2-mercaptoethanol), and 25 mL of heat inactivated fetal bovine serum (FBS) to 222.25 mL of Dulbelcco's Modified Eagle Medium (DMEM). F...

<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 nanoparticle vaccine platforms have shown efficacy when administered intranasally in single dose regimens5. Measuring the activation of the resident phagocytic cell populations in the lungs induced by this vaccine delivery platform permits evaluation of its potential capability to ultimately promote adaptive immune responses.
	Specifically, harvesting alveolar macrophages from lung lavage fluid and treating them with different formulations of the nanoparticles pr...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>cAM Media</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Cellgro</td>
			<td>15-013-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 mM 2-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148-25ML</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin 10,000 &mu;g/ mL Solution</td>
			<td>Cellgro</td>
			<td>30-002-CI</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>FACS Buffer</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>S671-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium phosphate</td>
			<td>Fisher Scientific</td>
			<td>MK7868500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Fisher Scientific</td>
			<td>P217500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium phosphate</td>
			<td>Fisher Scientific</td>
			<td>P288-200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BSA (Bovine Serum Albumin)</td>
			<td>Sigma</td>
			<td>A7888</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Antibodies</td>
		</tr>
		<tr>
			<td>Rat IgG</td>
			<td>Sigma</td>
			<td>I4341</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-Ms CD16/32</td>
			<td>eBioscience</td>
			<td>16-0161</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-Ms MHC II haplotype I-A/I-E, clone M5/114.15.2, conjugated to fluorescein isothiocyanate (FITC)</td>
			<td>eBioscience</td>
			<td>11-5321</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-mouse CD86, clone GL-1, conjugated to allophycocyanin (APC)-Cy7</td>
			<td>Biolegend</td>
			<td>105030</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-mouse CD40, clone 1C10, conjugated to APC</td>
			<td>eBioscience</td>
			<td>17-0401</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-mouse CD209, clone 5H10, conjugated to Biotin</td>
			<td>eBioscience</td>
			<td>13-2091</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11b, clone M1/70, conjugated to Alexa Fluor 700</td>
			<td>eBioscience</td>
			<td>56-0112</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-mouse F4/80, clone BM8, conjugated to phycoerythrin (PE)-Cy7</td>
			<td>eBioscience</td>
			<td>25-4801</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PE-Texas red conjugated Streptavidin</td>
			<td>BD Biosciences</td>
			<td>551487</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Other Supplies and Reagents</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>A405-20</td>
			<td>Used as 70% (v/v)</td>
		</tr>
		<tr>
			<td>Compressed CO2</td>
			<td>Linweld</td>
			<td>16000060</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 mL Syringe</td>
			<td>BD Biosciences</td>
			<td>309659</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sovereign 3 &frac12;" Fr Tom Catcatheter</td>
			<td>Kendall</td>
			<td>703021</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biosafety Cabinet</td>
			<td>NUAIRE</td>
			<td>Series 22</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dissection Scissors</td>
			<td>Fisher</td>
			<td>138082</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Roboz</td>
			<td>RS-8254</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS, 1X without calcium and magnesium</td>
			<td>Cellgro</td>
			<td>21-040-CM</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 mL Centrifuge Tubes with Screw Cap</td>
			<td>VWR International</td>
			<td>21008-216</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Six-well Tissue Culture Treated Plates</td>
			<td>Costar</td>
			<td>3516</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plastic Tube Racks</td>
			<td>Nalgene</td>
			<td>5970</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell Scraper 24 cm</td>
			<td>TPP</td>
			<td>99002</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5 mL Polystyrene Round-Bottom Tube</td>
			<td>Falcon</td>
			<td>352008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pipet-aid XL</td>
			<td>Drummond</td>
			<td>4-000-105</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10, 5, and 2 mL Pipettes</td>
			<td>Fisher</td>
			<td>13-675</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>200 and 10 &mu;L micropipettors</td>
			<td>Gilson Pipetman</td>
			<td>F123601</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>200 and 10 &mu;L pipette tips</td>
			<td>Fisher</td>
			<td>02-707</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BD Stabilizing Fixative</td>
			<td>BD Biosciences</td>
			<td>338036</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isoton II Diluent</td>
			<td>Beckman-Coulter</td>
			<td>8546719</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Zap-oglobin II Lytic Reagent</td>
			<td>Beckman-Coulter</td>
			<td>7546138</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Coulter Counter Polystyrene Vials</td>
			<td>Beckman-Coulter</td>
			<td>14310-684</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Test Tubes</td>
			<td>BD Biosciences</td>
			<td>352008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Refrigerated Centrifuge</td>
			<td>Labnet</td>
			<td>50075040</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Humidified Incubator CO2</td>
			<td>Nuaire</td>
			<td>Model Autoflow 8500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FACSCanto Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td>338960</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Coulter Particle Counter Z1</td>
			<td>Beckman-Coulter</td>
			<td>WS-Z1DUALPC</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sonicator Liquid Processing Equipment with Microtip</td>
			<td>Misonix</td>
			<td>Model No. S-4000</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles 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

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

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

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