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

Questo lavoro è stato sostenuto da NIH concedere R01-3155-53502 e Washington University-Pfizer accordo Ricerca Biomedica.

1. Animal preparazione: Questo protocollo utilizza adulto femmina adulta LysM-eGFP topi, nei quali neutrofili e macrofagi sono fluorescente mediante l&#39;espressione di eGFP 1. <ol class="lalpha"><li> L&#39;etichettatura dei vasi sanguigni: Per visualizzare i vasi sanguigni, 15μl di non mirato quantum dots (655nm) sono diluiti in 100 ml di PBS e iniettato per via endovenosa nel topo 10-30 minuti prima di imaging. </li><li> Anestesia: <ol><li> Posizionare il mouse nella ...

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	<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>

In questo protocollo video dimostriamo le modalità di imaging non invasiva 2P di reclutamento dei leucociti in risposta all&#39;infiammazione. La zampa è un sito classico fisiologici per lo studio dell&#39;infiammazione come la malattia di allergie, infezioni e autoimmuni 4-7. 2P di imaging fornisce un quadro dettagliato delle fasi distinte nei percorsi traffico dei leucociti, dal rotolamento e adesione nei vasi sanguigni di chemiotassi ai siti delle funzioni effettrici. Abbiamo...

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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>
<td>&nbsp;</td>
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<td>Quick gel</td>
<td>&nbsp;</td>
<td>Duro</td>
<td>&nbsp;</td>
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<td>Puralube Vet Ointment </td>
<td>&nbsp;</td>
<td>Pharmaderm Animal Health</td>
<td>&nbsp;</td>
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<td>BD</td>
<td>08290-3284-38</td>
<td>&nbsp;</td>
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non-invasive imaging of leukocyte homing and migration in vivo

A due fotoni (2P) microscopia è una tecnica di imaging ad alta risoluzione che è stata ampiamente adattato dai biologi. Il valore di microscopia 2P è che fornisce ricche informazioni spazio-temporali per quanto riguarda i comportamenti delle cellule all&#39;interno dei tessuti intatti e in topi vivi. Reclutamento dei leucociti svolgono un ruolo importante nella difesa dell&#39;ospite contro le infezioni e quando incontrollato, può contribuire a malattie infiammatorie e autoimmuni. Studiare il reclutamento dei leucociti in vivo è tecnicamente impegnativo poiché le cellule si spostano rapidamente all&#39;interno dei vasi situato in profondità all&#39;interno dei tessuti dispersione della luce. Fino ad oggi, gli studi di imaging più intravitale richiedono una preparazione chirurgica per esporre i vasi sanguigni e dei tessuti. Per evitare il danno ai tessuti e l&#39;infiammazione indotta da un intervento chirurgico se stessa, qui, si descrive un non-invasiva a cella singola approccio di imaging che può essere utilizzato per studiare il traffico dei leucociti nella zampa del mouse e falangi. Discutiamo gli aspetti tecnici della nostra preparazione 2P imaging e camminare il lettore attraverso un tipico esperimento di configurazione iniziale di esecuzione e di raccolta dei dati.

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

Qui, descriviamo un non-invasiva a due fotoni (2P) approccio microscopia per lo studio dei leucociti homing nella zampa del mouse. Discutiamo gli aspetti tecnici della nostra preparazione dei tessuti di imaging e camminare il lettore attraverso un tipico esperimento di configurazione iniziale di esecuzione e di raccolta dei dati.

Non invasiva di imaging di homing dei leucociti e la migrazione 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

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

Ricerca

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Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. ...

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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 ...

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

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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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Imaging non invasiva della risposta immunitaria innata in un modello di Zebrafish larvale<em&gt; Streptococcus iniae</em&gt; Infezione

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 ...

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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

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