Cette étude a été financée par la Deutsche Forschungsgemeinschaft à SW (DFG WA 2804) et une bourse universitaire pour SW à partir du Centre de recherche expérimentale et clinique, une coopération du Centre Max Delbrück de médecine moléculaire et de la faculté de médecine Charité à Berlin. Les bailleurs de fonds n&#39;ont joué aucun rôle dans la conception de l&#39;étude, la collecte de données et d&#39;analyse, la décision de publier ou de la préparation du manuscrit. Nous remercions M. Robert Westphal pour le support technique lors de son stage dans notre laboratoire.

<ol>
	<li>Yeh, T. C., Zhang, W., Ildstad, S. T., Ho, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+dynamic+MRI+tracking+of+rat+T-cells+labeled+with+superparamagnetic+iron-oxide+particles.">In vivo dynamic MRI tracking of rat T-cells labeled with superparamagnetic iron-oxide particles.</a> Magn. Reson. Med. 33 (2), 200-208 (1995).</li><li>Weissleder, R., Cheng, H. C., Bogdanova, A., Bogdanov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetically+labeled+cells+can+be+detected+by+MR+imaging.">Magnetically labeled cells can be detected by MR imaging.</a> J. Magn. Reson. Imaging. 7 (1), 258-263 (1997).</li><li>Franklin, R. J., Blaschuk, K. L., Bearchell, M. C., Prestoz, L. L., Setzu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+of+transplanted+oligodendrocyte+precursors+in+the+rat+brain.">Magnetic resonance imaging of transplanted oligodendrocyte precursors in the rat brain.</a> NeuroReport. 10 (18), 3961-3965 (1999).</li><li>de Vries, I. J., Lesterhuis, W. J., Barentsz, J. O., Verdijk, P., van Krieken, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+tracking+of+dendritic+cells+in+melanoma+patients+for+monitoring+of+cellular+therapy.">Magnetic resonance tracking of dendritic cells in melanoma patients for monitoring of cellular therapy.</a> Nat. Biotechnol. 23 (11), 1407-1413 (2005).</li><li>Liu, W., Frank, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+quantification+of+magnetically+labeled+cells+by+cellular+MRI.">Detection and quantification of magnetically labeled cells by cellular MRI.</a> Eur. J. Radiol. 70 (2), 258-264 (2009).</li><li>Ahrens, E. T., Flores, R., Xu, H., Morel, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+platform+for+tracking+immunotherapeutic+cells.">In vivo imaging platform for tracking immunotherapeutic cells.</a> Nat. Biotechnol. 23 (8), 983-987 (2005).</li><li>Holland, G. N., Bottomley, P. A., Hinshaw, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F-19+Magnetic-Resonance+Imaging.">F-19 Magnetic-Resonance Imaging.</a> Journal of Magnetic Resonance. 28 (1), 133-136 (1977).</li><li>Partlow, K. C., Chen, J., Brant, J. A., Neubauer, A. M., Meyerrose, T. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=19F+magnetic+resonance+imaging+for+stem/progenitor+cell+tracking+with+multiple+unique+perfluorocarbon+nanobeacons.">19F magnetic resonance imaging for stem/progenitor cell tracking with multiple unique perfluorocarbon nanobeacons.</a> FASEB J. 21 (8), 1647-1654 (2007).</li><li>Ruiz-Cabello, J., Walczak, P., Kedziorek, D. A., Chacko, V. P., Schmieder, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+"hot+spot"+MR+imaging+of+neural+stem+cells+using+fluorinated+nanoparticles.">In vivo "hot spot" MR imaging of neural stem cells using fluorinated nanoparticles.</a> Magn. Reson. Med. 60 (6), 1506-1511 (2008).</li><li>Srinivas, M., Morel, P. A., Ernst, L. A., Laidlaw, D. H., Ahrens, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorine-19+MRI+for+visualization+and+quantification+of+cell+migration+in+a+diabetes+model.">Fluorine-19 MRI for visualization and quantification of cell migration in a diabetes model.</a> Magn. Reson. Med. 58 (4), 725-734 (2007).</li><li>Waiczies, H., Lepore, S., Janitzek, N., Hagen, U., Seifert, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfluorocarbon+particle+size+influences+magnetic+resonance+signal+and+immunological+properties+of+dendritic+cells.">Perfluorocarbon particle size influences magnetic resonance signal and immunological properties of dendritic cells.</a> PLoS ONE. 6 (7), e21981 (2011).</li><li>Matheu, M. P., Sen, D., Cahalan, M. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Bone+Marrow+Derived+Murine+Dendritic+Cells+for+Use+in+2-photon+Imaging.">Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging.</a> J. Vis. Exp. (17), e773 (2008).</li><li>Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+large+numbers+of+dendritic+cells+from+mouse+bone+marrow+cultures+supplemented+with+granulocyte/macrophage+colony-stimulating+factor.">Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.</a> Journal of Experimental Medicine. 176 (6), 1693-1702 (1992).</li><li>Lanzavecchia, A., Sallusto, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ralph+M.+Steinman.+1943-2011.">Ralph M. Steinman. 1943-2011.</a> Cell. 147 (6), 1216-1217 (1943).</li><li>Srinivas, M., Turner, M. S., Janjic, J. M., Morel, P. A., Laidlaw, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cytometry+of+antigen-specific+t+cells+using+19F.">In vivo cytometry of antigen-specific t cells using 19F.</a> MRI. Magn. Reson. Med. 62 (3), 747-753 (2009).</li><li>Srinivas, M., Heerschap, A., Ahrens, E. T., Figdor, C. G., de Vries, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=(19)F+MRI+for+quantitative+in+vivo+cell+tracking.">(19)F MRI for quantitative in vivo cell tracking.</a> Trends Biotechnol. 28 (7), 363-370 (2010).</li><li>Lammermann, T., Bader, B. L., Monkley, S. J., Worbs, T., Wedlich-Soldner, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+leukocyte+migration+by+integrin-independent+flowing+and+squeezing.">Rapid leukocyte migration by integrin-independent flowing and squeezing.</a> Nature. 453 (7191), 51-55 (2008).</li><li>Liu, M. S., Long, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfluoroctylbromide+as+a+diagnostic+contrast+medium+in+gastroenterography.">Perfluoroctylbromide as a diagnostic contrast medium in gastroenterography.</a> Radiology. 122 (1), 71-76 (1977).</li><li>Kwiatkowska, K., Sobota, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+pathways+in+phagocytosis.">Signaling pathways in phagocytosis.</a> Bioessays. 21 (5), 422-431 (1999).</li><li>Mitragotri, S., Lahann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+approaches+to+biomaterial+design.">Physical approaches to biomaterial design.</a> Nat. Mater. 8 (1), 15-23 (2009).</li><li>Hoult, D. I., Richards, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+signal-to-noise+ratio+of+the+nuclear+magnetic+resonance+experiment.">The signal-to-noise ratio of the nuclear magnetic resonance experiment.</a> J. Magn. Reson. 24 (1), 71-85 (1976).</li><li>Kovacs, H., Moskau, D., Spraul, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryogenically+cooled+probes+-+a+leap+in+NMR+technology.">Cryogenically cooled probes - a leap in NMR technology.</a> Prog. Nucl. Magn. Reson. Spectrosc. 46 (2-3), 131-155 (2005).</li><li>Waiczies, H., Millward, J. M., Lepore, S., Infante-Duarte, C., Pohlmann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Cellular+Infiltrates+during+Early+Stages+of+Brain+Inflammation+with+Magnetic+Resonance+Microscopy.">Identification of Cellular Infiltrates during Early Stages of Brain Inflammation with Magnetic Resonance Microscopy.</a> PLoS ONE. 7 (3), e32796 (2012).</li><li>Haacke, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Magnetic resonance imaging physical principles and sequence design. , (1999).</li></ol>

Les auteurs déclarent n&#39;avoir aucun conflit d&#39;intérêts financiers.

Cette méthode d&#39;employer 19 F / 1 H IRM pour suivre le mouvement de DC dans le ganglion lymphatique donne l&#39;occasion d&#39;étudier les schémas de migration des cellules immunitaires in vivo. Les cellules dendritiques sont d&#39;excellents exemples de la migration rapide des cellules immunitaires qui sont capables de manœuvrer à travers des structures tridimensionnelles sans bien adhérentes à des substrats spécifiques 17. Bien que la faible résolution spatiale (?...

The tracking of cells in vivo is a crucial aspect in several fields of biomedicine. For this, noninvasive imaging techniques that can selectively localize cells over a period of time are extremely valuable. Prior to the development of three-dimensional magnetic resonance imaging (MRI), the tracking of immune cell migration was limited to microscopic analyses or tissue biopsies. Cell tracking with the help of MRI has gained immense attention in the past few years, not only for immunologists studying immune cell behavior in vivo, but also for clinical and stem cell researchers. During the mid-90s, the first studies on iron oxide nanoparticles 1 initiated a cascade of developments for tracking cells with MRI. Iron oxide particles shorten the MR relaxation time (T2*) of the labeled cells and thus cause signal depletion in MR images. Iron oxide particles have been employed to label macrophages 2, oligodendrocyte progenitors 3 and many other cell types. Some of these particles have also been clinically approved by the FDA for labeling cellular vaccines in melanoma patients 4. Since in vivo or ex vivo labeling of cells with iron oxide particles relies on a shortening of the T2* signal and the latter could be also brought about by in vivo susceptibility-related T2* effects such as micro bleeds, iron deposits or air bubbles, it might be difficult to identify labeled cells in vivo from other background T2* signal extinctions 5.
In this article, we describe a technique for tracking dendritic cells (DC) in vivo by employing 19F/1H magnetic resonance imaging (MRI). This cell tracking technology was only introduced in 2005 6, several years after the first recognized applications for 19F in MRI had been reported 7. One important advantage of 19F over iron oxide particle cell labeling is the low biological occurrence of 19F in tissue; this makes it possible to track cells very selectively with basically background-free images. Furthermore, it is possible to overlay the 19F MR signal from the transplanted labeled cells with anatomical images obtained from conventional 1H MRI. 19F/1H MRI is therefore considerably relevant for studies investigating cell migration in vivo. Cells studied with this method are labeled with 19F-rich particles. Synthetically-derived perfluorocarbons (PFCs) consisting primarily of carbon and fluorine atoms are commonly used to prepare the particles. These compounds are insoluble in water and need to be emulsified prior to application in vitro or in vivo. The usual size of the PFC particles that have been employed by other groups for in vivo 19F-MRI tracking experiments ranges between 100 nm and 245 nm 6,8-10. We have however shown that the efficiency in labeling dendritic cells with perfluoro-15-crown-5-ether (PFCE) particles increases with increasing particle size (&gt;560 nm).11

<table border="1"><tbody><tr><td></td><td></td><td></td><td> REACTIFS </td></tr><tr><td> Souris C57BL / 6 </td><td> Charles River, Berlin </td><td></td><td></td></tr><tr><td> RPMI </td><td> Gibco </td><td> 21875-091 </td><td></td></tr><tr><td> FBS supérieure </td><td> Biochrom AG </td><td> S 0615 </td><td></td></tr><tr><td> HEPES </td><td> Gibco Invitrogen- </td><td> 15630-056 </td><td></td></tr><tr><td> Pénicilline-streptomycine </td><td> Gibco </td><td> 15140-122 </td><td></td></tr><tr><td> L-glutamine </td><td> Gibco </td><td> 25030-024 </td><td></td></tr><tr><td> PBS Dulbecco </td><td> Sigma Aldrich </td><td> D8662 </td><td></td></tr><tr><td> PFA </td><td> Santa Cruz </td><td> sc-281692 </td><td></td></tr><tr><td> Perfluoro-15-couronne-5-éther </td><td> ChemPur </td><td> 391 -1996 </td><td></td></tr><tr><td> Pluronic F-68 </td><td> Sigma Aldrich </td><td> P5556 </td><td></td></tr><tr><td> Des boîtes de Pétri (35 x 10 mm) </td><td> VWR, Allemagne </td><td> 391-1996 </td><td></td></tr><tr><td> 27 ½ seringues G </td><td> VWR, Allemagne </td><td> 612-0151 </td><td></td></tr><tr><td> Tamis en nylon de cellules um (100 mesh) </td><td> VWR, Allemagne </td><td> 734-0004 </td><td></td></tr><tr><td> RMN tubes </td><td> VWR, Allemagne </td><td> 634-0461 </td><td></td></tr><tr><td></td><td></td><td></td><td> ÉQUIPEMENT </td></tr><tr><td> Outils de dissection </td><td> FST </td><td></td><td></td></tr><tr><td> Incubateur à CO 2 </td><td> Liant </td><td></td><td></td></tr><tr><td> Petit animal MR système </td><td> Bruker BioSpin </td><td></td><td> 9.4T BioSpec 94/20 USR,Acquisition ParaVision et de logiciels de traitement </td></tr><tr><td> 1 H / 19 F à double bobine RF accordable de volume </td><td> Rapid Biomed, Würzburg, Allemagne </td><td></td><td> 35 mm de diamètre intérieur, 50 mm de longueur </td></tr><tr><td> 19 bobine spectroscopie F </td><td> interne </td><td></td><td> air / match bobine en boucle, 4 tours, diamètre intérieur 5 mm, 10 mm de long, deux condensateurs pour le réglage et l&#39;appariement </td></tr><tr><td> Système d&#39;inhalation d&#39;isoflurane </td><td> Föhr Medical Instruments GmbH </td><td></td><td></td></tr><tr><td> Modèle du système de surveillance des animaux 1025 </td><td> SA Instruments Inc, New York, USA </td><td></td><td></td></tr></tbody></table>

monitoring dendritic cell migration using 19f / 1h magnetic resonance imaging

Progrès continus en matière modalités d&#39;imagerie non invasives comme l&#39;imagerie par résonance magnétique (IRM) ont grandement amélioré notre capacité à étudier les processus physiologiques ou pathologiques dans les organismes vivants. IRM s&#39;avère également être un outil précieux pour la capture des cellules transplantées in vivo. Les premières stratégies de marquage cellulaire pour utilisation d&#39;agents de contraste IRM qui influencent les temps de relaxation MR (T1, T2, T2 *) et conduire à une amélioration (T1) ou l&#39;épuisement (T2 *) du signal où les cellules marquées sont présentes. T2 agents d&#39;amélioration * tels que les agents ultrapetites d&#39;oxyde de fer (USPIO) ont été utilisés pour étudier la migration cellulaire et certains ont également été approuvé par la FDA pour une application clinique. Un inconvénient de T2 * agents est la difficulté de distinguer l&#39;extinction du signal créé par les cellules marquées d&#39;autres artefacts tels que des caillots de sang, des saignements ou des micro-bulles d&#39;air. Dans cet article, nous décrivons une nouvelle technique pour le suivi in vivo des cellules quisont basées sur le marquage des cellules avec du fluor (19 F)-particules riches. Ces particules sont préparées par émulsification d&#39;hydrocarbures perfluorés (PFC) et de composés ensuite utilisé pour marquer les cellules, qui peuvent ensuite être visualisées par 19 F IRM. Les avantages importants de PFC pour le suivi des cellules in vivo comprennent (i) l&#39;absence de carbone lié à 19 F in vivo, ce qui donne alors des images sans fond et complète de cellules selectivityand (ii) la possibilité de quantifier le signal de la cellule de 19 F spectroscopie MR .

Dix-huit à vingt et un heures suivant l&#39;application intradermique, 19 F-étiquetés cellules dendritiques (DC) migrent dans le ganglion lymphatique poplité drainant. Le mouvement des DC via les canaux lymphatiques dans le ganglion drainant politeal peut être apprécié par superposition des images 1 H anatomiques avec les 19 images DC F (figure 2A). Nous avons déjà fait état ​​de la migration de ces cellules in vivo, ainsi que l&#39;impact de la <s...

Watch this Scientific Journal Video about Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging at JoVE.com

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Suivi des cellules en utilisant l&#39;IRM a attiré l&#39;attention remarquable dans les dernières années. Ce protocole décrit le marquage des cellules dendritiques avec le fluor (<sup&gt; 19</sup&gt; Particules F)-riches, l&#39;application in vivo de ces cellules, et la surveillance de l&#39;étendue de leur migration vers le ganglion drainant avec<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H IRM et<sup&gt; 19</sup&gt; F MRS.

La migration des cellules dendritiques de surveillance utilisant l&#39;imagerie par résonance 19F / 1H magnétique

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

monitoring-dendritic-cell-migration-using-19f-1h-magnetic-resonance

Research

JoVE Journal

Immunology and Infection

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

11.7K vues.  A joint cooperation between the Charité Medical Faculty and the Max Delbrück Center for Molecular Medicine. Suivi des cellules en utilisant l'IRM a attiré l'attention remarquable dans les dernières années. Ce protocole décrit le marquage des cellules dendritiques avec le fluor ( 19 Particules F)-riches, l'application in vivo de ces cellules, et la surveillance de l'étendue de leur migration vers le ganglion drainant avec 19 F / 1 H IRM et 19 F MRS.

Vidéo: Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

L&#39;imagerie non invasive des Homing leucocytes et des migrations In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Recherche

Immunologie et infection

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

Imagerie en temps réel des leucotriènes B 4</subMigration> Cell Mediated et BLT1 Interactions avec β-arrestine

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

Les analyses concurrentielles Homing d&#39;étude Gut tropisme migration des lymphocytes T

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

Protocole optimisé pour une transfection efficace des cellules dendritiques, sans maturation cellulaire

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Préparation de l&#39;échantillon de<em&gt; Mycobacterium tuberculosis</em&gt; Extraits pour Magnétique Nucléaire études métabolomiques résonance

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

L&#39;analyse de la maturation des cellules dendritiques pulmonaire et de la migration au cours de l&#39;inflammation allergique des voies respiratoires

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

Génération d’un nouveau vaccin à cellules dendritiques utilisant des cellules souches de mélanome et de cancer malpighien

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

Imagerie CD4 T Cellule interstitielle Migration dans les Derme Enflammé

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with &lt;em&gt;Mycobacterium bovis&lt;/em&gt; Bacille Calmette-Gu&amp;#233;rin

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

Un test basé CFSE pour étudier la migration de la peau murine cellules dendritiques dans les ganglions lymphatiques de drainage lors de l&#39;infection avec<em&gt; Mycobacterium bovis</em&gt; Bacille Calmette-Guérin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Génération d’un grand nombre de progéniteurs myéloïdes et précurseurs des cellules dendritiques de murin la moelle osseuse à l’aide d’une nouvelle cellule stratégie de tri

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...