The authors would like to thank the ONR-MURI Award (NN00014-06-1-1176) and the U.S. Army Medical Research and Materiel Command (Grant Numbers W81XWH-09-1-0386 and W81XWH-10-1-0806) for financial support.

1. RAW 264.7 Cell Culture
<ol>
	<li>Harvest RAW 264.7 cells from their flasks when they reach confluency by scraping them gently with a cell scraper. Count and plate them into a 24-well cell culture dish at a density of 5 x 105 cells/well in 0.5 mL complete Dulbecco's Modified Eagle Medium (cDMEM; 10% heat-inactivated fetal bovine serum (FBS), 2 mM Glutamax, and 10 mM HEPES) and incubate overnight at 37 &deg;C in a 5% CO2 incubator.</li>
</ol>
2. Pathogenic Salmonella ent...

<ol>
	<li>Ulery, B. D., Kumar, D., Ramer-Tait, A. E., Metzger, D. W., 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=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>Kasturi, S. P., Skountzou, I., Albrecht, R. A., Koutsonanos, D., Hua, T., Nakaya, H. I., Ravindran, R., Stewart, S., Alam, M., Kwissa, M., Villinger, F., Murthy, N., Steel, J., Jacob, J., Hogan, R. J., Garc&#237;a-Sastre, A., Compans, R., Pulendran, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programming+the+magnitude+and+persistence+of+antibody+responses+with+innate+immunity.">Programming the magnitude and persistence of antibody responses with innate immunity.</a> Nature. 470, 543-547 (2011).</li><li>Rice-Ficht, A. C., Arenas-Gamboa, A. M., Kahl-McDonagh, M. M., Ficht, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymeric+particles+in+vaccine+delivery.">Polymeric particles in vaccine delivery.</a> Curr. Opin. Microbiol. 13, 106-112 (2010).</li><li>Jain, J. P., Chitkara, D., Kumar, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyanhydrides+as+localized+drug+delivery+carrier:+an+update.">Polyanhydrides as localized drug delivery carrier: an update.</a> Expert. Opin. Drug. Deliv. 5, 889-907 (2008).</li><li>Pfeifer, B. A., Burdick, J. A., Little, S. R., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(ester-anhydride):poly(beta-amino+ester)+micro-+and+nanospheres:+DNA+encapsulation+and+cellular+transfection.">Poly(ester-anhydride):poly(beta-amino ester) micro- and nanospheres: DNA encapsulation and cellular transfection.</a> Int. J. Pharm. 304, 210-219 (2005).</li><li>Ahmed, F., Friend, S., George, T. C., Barteneva, N., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numbers+matter:+quantitative+and+dynamic+analysis+of+the+formation+of+an+immunological+synapse+using+imaging+flow+cytometry.">Numbers matter: quantitative and dynamic analysis of the formation of an immunological synapse using imaging flow cytometry.</a> J. Immunol. Methods. 347, 79-86 (2009).</li><li>Hampton, M. B., Winterbourn, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+quantifying+phagocytosis+and+bacterial+killing+by+human+neutrophils.">Methods for quantifying phagocytosis and bacterial killing by human neutrophils.</a> J. Immunol. Methods. 232, 15-22 (1999).</li><li>Rieger, A. M., Hall, B. E., Barreda, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+activation+differentially+modulates+particle+binding,+phagocytosis+and+downstream+antimicrobial+mechanisms.">Macrophage activation differentially modulates particle binding, phagocytosis and downstream antimicrobial mechanisms.</a> Dev. Comp. Immunol. 34, 1144-1159 (2010).</li><li>Murphy, K. C., Campellone, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lambda+Red-mediated+recombinogenic+engineering+of+enterohemorrhagic+and+enteropathogenic+E.+coli.">Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli.</a> BMC. Mol. Biol. 4, 11 (2003).</li><li>Karsi, A., Lawrence, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broad+host+range+fluorescence+and+bioluminescence+expression+vectors+for+Gram-negative+bacteria.">Broad host range fluorescence and bioluminescence expression vectors for Gram-negative bacteria.</a> Plasmid. 57, 286-295 (2007).</li><li>Ulery, B. D., Phanse, Y., Sinha, A., Wannemuehler, M. J., Narasimhan, B., Bellaire, B. H. <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>Doherty, G. J., McMahon, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+endocytosis.">Mechanisms of endocytosis.</a> Annu. Rev. Biochem. 78, 857-902 (2009).</li><li>Vercauteren, D., Vandenbroucke, R. E., Jones, A. T., Rejman, J., Demeester, J., De Smedt, S. C., Sanders, N. N., Braeckmans, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+inhibitors+to+study+endocytic+pathways+of+gene+carriers:+optimization+and+pitfalls.">The use of inhibitors to study endocytic pathways of gene carriers: optimization and pitfalls.</a> Mol. Ther. 18, 561-569 (2010).</li><li>Di Marzio, L., Marianecci, C., Cinque, B., Nazzarri, M., Cimini, A. M., Cristiano, L., Cifone, M. G., Alhaique, F., Carafa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH-sensitive+non-phospholipid+vesicle+and+macrophage-like+cells:+binding,+uptake+and+endocytotic+pathway.">pH-sensitive non-phospholipid vesicle and macrophage-like cells: binding, uptake and endocytotic pathway.</a> Biochim. Biophys. Acta. 1778, 2749-2756 (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></ol>

Sherree L. Friend is employed by Amnis Corporation, which manufactures the ImageStreamX system.

Studies have shown that biodegradable nanoparticles based on poly(lactic-co-glycolic acid (PLGA) or polyanhydrides can be used to deliver encapsulated antigens or drugs to target cells. Uptake of these nanoparticles by phagocytic cells is important for their effectiveness, thus making quantitative analysis of internalization critical in designing novel nanoparticle delivery systems. By using this method, differential uptake of nanoparticles by various cell types can be analyzed with ease. To date, conventional microscopy...

<table border="1">
	 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>RAW 264.7 cell line</td>
 <td>American Type Culture Collection (ATCC)</td>
 <td>TIB-71</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dulbecco's Modified Eagle Medium (DMEM)</td>
 <td>Cellgro</td>
 <td>10-013-CV</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal bovine serum</td>
 <td>Atlanta Biologicals</td>
 <td>S 11150</td>
 <td>Premium Grade</td>
 </tr>
 <tr>
 <td>Glutamax</td>
 <td>Gibco</td>
 <td>35050-061</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Gibco</td>
 <td>15630-080</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>24-well plate</td>
 <td>TPP</td>
 <td>92024</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cell culture Flasks</td>
 <td>TPP</td>
 <td>90151</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cell scraper</td>
 <td>TPP</td>
 <td>99002</td>
 <td>24 cm</td>
 </tr>
 <tr>
 <td>Salmonella entericaserovar Typhimurium</td>
 <td>ATCC</td>
 <td>14028</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>BTX ECM630 Electro Cell Manipulator</td>
 <td>BTX Harvard Apparatus</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MOPS</td>
 <td>Fisher Scientific</td>
 <td>BP308</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phosphate buffered saline (PBS)</td>
 <td>Cellgro</td>
 <td>21-040-CV</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ultrasonic liquid processor</td>
 <td>Misonix</td>
 <td>S-4000</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cytochalasin-D</td>
 <td>Sigma-Aldrich,</td>
 <td>C8273</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Formaldehyde</td>
 <td>Polysciences</td>
 <td>04018</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Wash buffer</td>
 <td>2% heat inactivated FBS, 0.1% sodium azide in PBS.</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Perm/wash buffer</td>
 <td>BD Biosciences</td>
 <td>554714</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Clear-view snap cap microtubes</td>
 <td>Sigma</td>
 <td>T4816</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Alexa Fluor phalloidin 660</td>
 <td>Invitrogen</td>
 <td>A22285</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>ImageStreamX</td>
 <td>Amnis Corporation</td>
 <td>100200</td>
 <td>Options: 658nm laser, autosampler</td>
 </tr>
 <tr>
 <td>Sodium azide</td>
 <td>Fisher Scientific</td>
 <td>S 227I-500</td>
 <td>&nbsp;</td>
 </tr>
	 </tbody>
 </table>

analyzing cellular internalization of nanoparticles and bacteria by multi-spectral imaging flow cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

Watch this Scientific Journal Video about Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry at JoVE.com

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

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

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

analyzing-cellular-internalization-nanoparticles-bacteria-multi

Research

JoVE Journal

Bioengineering

16.2K vues.  Iowa State University. Dans cet article, nous décrivons une méthode utilisant multi-spectrale cytométrie de flux d'imagerie pour quantifier l'internalisation des nanoparticules ou des bactéries polyanhydride par des cellules RAW 264.7.