Name: non-invasive imaging of leukocyte homing and migration in vivo
Uploaded: 2010-12-05T17:10:00
Description: Watch this Scientific Journal Video about Non-invasive Imaging of Leukocyte Homing and Migration in vivo at JoVE.com

Ce travail a été soutenu par NIH R01-3155-53502 et de l&#39;Université de Washington-Pfizer accord de recherche biomédicale.

1. La préparation des animaux: Ce protocole utilise adulte femelle adulte LysM-eGFP souris, dans lequel les neutrophiles et les macrophages sont marqués par fluorescence par l&#39;expression de eGFP 1. <ol class="lalpha"><li> Étiquetage des vaisseaux sanguins: Pour visualiser les vaisseaux sanguins, 15μl de points quantiques non-ciblées (655nm) sont dilués dans 100 ul de PBS et injecté par voie intraveineuse chez la souris 10-30 min avant l&#39;imagerie. </li><li> ...

<ol>
	<li>Faust, N., Varas, F., Kelly, L. M., Heck, S., Graf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertion+of+enhanced+green+fluorescent+protein+into+the+lysozyme+gene+creates+mice+with+green+fluorescent+granulocytes+and+macrophages.">Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages.</a> Blood. 96, 719-726 (2000).</li><li>Zinselmeyer, B. H., Lynch, J. N., Zhang, X., Aoshi, T., Miller, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Video-rate+two-photon+imaging+of+mouse+footpad+-+a+promising+model+for+studying+leukocyte+recruitment+dynamics+during+inflammation.">Video-rate two-photon imaging of mouse footpad - a promising model for studying leukocyte recruitment dynamics during inflammation.</a> Inflamm Res. 57, 93-96 (2008).</li><li>Zinselmeyer, B. H., Dempster, J., Wokosin, D. L., Cannon, J. J., Pless, R., Parker, I., Miller, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+16.+Two-photon+microscopy+and+multidimensional+analysis+of+cell+dynamics.">Chapter 16. Two-photon microscopy and multidimensional analysis of cell dynamics.</a> Methods Enzymol. 461, 349-378 (2009).</li><li>Gray, D. F., Jennings, 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=Allergy+in+experimental+mouse+tuberculosis.">Allergy in experimental mouse tuberculosis.</a> Am Rev Tuberc. 72, 171-195 (1955).</li><li>Lin, A., Loughman, J. A., Zinselmeyer, B. H., Miller, M. J., Caparon, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptolysin+S+inhibits+neutrophil+recruitment+during+the+early+stages+of+Streptococcus+pyogenes+infection.+Infect+Immun.">Streptolysin S inhibits neutrophil recruitment during the early stages of Streptococcus pyogenes infection. Infect Immun.</a> 77, 5190-5201 (2009).</li><li>Smith, C. J., Zhang, Y., Koboldt, C. M., Muhammad, J., Zweifel, B. S., Shaffer, A., Talley, J. J., Masferrer, J. L., Seibert, K., Isakson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+analysis+of+cyclooxygenase-1+in+inflammation.">Pharmacological analysis of cyclooxygenase-1 in inflammation.</a> Proc Natl Acad Sci U S A. 95, 13313-13318 (1998).</li><li>Wipke, B. T., Allen, 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=Essential+role+of+neutrophils+in+the+initiation+and+progression+of+a+murine+model+of+rheumatoid+arthritis.">Essential role of neutrophils in the initiation and progression of a murine model of rheumatoid arthritis.</a> J Immunol. 167, 1601-1608 (2001).</li><li>Baez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+open+cremaster+muscle+preparation+for+the+study+of+blood+vessels+by+in+vivo+microscopy.">An open cremaster muscle preparation for the study of blood vessels by in vivo microscopy.</a> Microvasc Res. 5, 384-394 (1973).</li><li>Mempel, T. R., Scimone, M. L., Mora, J. R., von Andrian, U. H. <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+of+leukocyte+trafficking+in+blood+vessels+and+tissues.">in vivo imaging of leukocyte trafficking in blood vessels and tissues.</a> Curr Opin Immunol. 16, 406-417 (2004).</li><li>Miller, M. J., Wei, S. H., 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=Autonomous+T+cell+trafficking+examined+in+vivo+with+intravital+two-photon+microscopy.">Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy.</a> Proc Natl Acad Sci U S A. 100, 2604-2609 (2003).</li></ol>

Dans ce protocole vidéo nous montrent les procédures d&#39;imagerie non-invasive 2P du recrutement des leucocytes en réponse à l&#39;inflammation. Le coussinet plantaire est un site classique pour l&#39;étude de l&#39;inflammation physiologiques telles que la maladie d&#39;allergie, d&#39;infection et auto-immunes 4-7. 2P imagerie fournit une image détaillée des étapes distinctes dans les voies de trafic de leucocytes, de laminage et d&#39;adhérence dans les vaisseaux san...

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<td>Isoflurane, USP</td>
<td>&nbsp;</td>
<td>Butler Animal Health Supply</td>
<td>029405</td>
<td>&nbsp;</td>
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<td>655nm non-targeted quantum dots</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>Q21021MP</td>
<td>&nbsp;</td>
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<td>Tetramathylrhodamine conjugated E. coli BioParticles </td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>E2862</td>
<td>&nbsp;</td>
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<td>Listeria monocytogenes (Lm)</td>
<td>&nbsp;</td>
<td>Reference 2</td>
<td>&nbsp;</td>
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<tr>
<td>Quick gel</td>
<td>&nbsp;</td>
<td>Duro</td>
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<td>Puralube Vet Ointment </td>
<td>&nbsp;</td>
<td>Pharmaderm Animal Health</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Insulin syringes</td>
<td>&nbsp;</td>
<td>BD</td>
<td>08290-3284-38</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Thermo Scirntific</td>
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non-invasive imaging of leukocyte homing and migration in vivo

À deux photons (2P) est une technique de microscopie à haute résolution d&#39;imagerie qui a été largement adaptée par les biologistes. La valeur de la microscopie 2P est qu&#39;il fournit de riches informations sur les comportements spatio-temporelle des cellules dans les tissus intacts et dans des souris vivantes. Le recrutement des leucocytes jouent un rôle important dans la défense de l&#39;hôte contre l&#39;infection et quand incontrôlée, peut contribuer à des maladies inflammatoires et autoimmunes. Etudier le recrutement des leucocytes in vivo est un défi technique, car les cellules se déplacent rapidement dans les vaisseaux situés en profondeur dans les tissus diffusion de la lumière. À ce jour, la plupart des études d&#39;imagerie intravitale nécessitent une préparation chirurgicale pour exposer les vaisseaux sanguins et des tissus. Pour éviter les dommages tissulaires et l&#39;inflammation induite par la chirurgie elle-même, ici, nous décrivons une approche non invasive d&#39;imagerie à cellule unique qui peut être utilisée pour étudier le trafic des leucocytes dans le coussinet plantaire de souris et des phalanges. Nous discutons des aspects techniques de notre préparation imagerie 2P et marcher le lecteur à travers une expérience typique de la configuration initiale à la collecte de l&#39;exécution et des données.

Watch this Scientific Journal Video about Non-invasive Imaging of Leukocyte Homing and Migration in vivo at JoVE.com

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Ici, nous décrivons un non-invasive à deux photons approche microscopique (2P) pour l&#39;étude des leucocytes homing dans le coussinet plantaire de la souris. Nous discutons des aspects techniques de notre préparation imagerie des tissus et de marcher le lecteur à travers une expérience typique de la configuration initiale à la collecte de l&#39;exécution et des données.

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 ...

Research

JoVE Journal

Immunology and Infection

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Competitive Homing Assays to Study Gut-tropic T Cell Migration

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 ...

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Immunologie et infection

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Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

Non invasive d&#39;imagerie optique du système vasculaire lymphatique d&#39;une souris

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

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Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

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Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

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Imagerie non invasive de la réponse immunitaire innée dans un modèle de poisson zèbre larves de<em&gt; Streptococcus iniae</em&gt; Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

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Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

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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 ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

Non invasif<em&gt; In Vivo</em&gt; Imagerie optique à fluorescence d&#39;une activité MMP inflammatoire à l&#39;aide d&#39;un agent d&#39;imagerie fluorescente activable

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...