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

Dit werk werd ondersteund door NIH subsidie ​​R01-3155 tot 53.502 en de Universiteit van Washington-Pfizer Biomedical Research overeenkomst.

1. Dier voorbereiding: Dit protocol maakt gebruik van volwassen vrouwelijke volwassen LysM-eGFP muizen, waarin de neutrofielen en macrofagen fluorescent zijn gelabeld door de expressie van eGFP 1. <ol class="lalpha"><li> Labeling bloedvaten: Om dit te visualiseren bloedvaten, 15μl van de niet-gerichte quantum dots (655nm) worden verdund in 100 pi PBS en intraveneus geïnjecteerd in de muis 10-30 min. voor beeldvorming. </li><li> Anesthesie: <ol><li> Plaats de muis in de ...

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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 deze video protocol tonen we de procedures voor niet-invasieve beeldvorming van 2P leukocyten rekrutering in reactie op de ontsteking. De voetzool is een klassieke fysiologische site voor het bestuderen van ontsteking, zoals allergie, infectie en auto-immuunziekte 4-7. 2P beeldvorming geeft een gedetailleerd beeld van de verschillende stappen in leukocyten mensenhandel wegen, uit te rollen en de hechting in de bloedvaten te chemotaxis naar sites van effector functie. We hebben ...

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		<th>Catalogue Number</th>
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<td>Isoflurane, USP</td>
<td>&nbsp;</td>
<td>Butler Animal Health Supply</td>
<td>029405</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>
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<td>Listeria monocytogenes (Lm)</td>
<td>&nbsp;</td>
<td>Reference 2</td>
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<td>Quick gel</td>
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<td>Duro</td>
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<td>Puralube Vet Ointment </td>
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non-invasive imaging of leukocyte homing and migration in vivo

Twee-foton (2P) microscopie is een hoge resolutie imaging techniek die heeft over het algemeen aangepast door biologen. De waarde van 2P microscopie is dat het rijk spatiotemporele informatie met betrekking tot het gedrag binnen de cel intact weefsels en in levende muizen biedt. Leukocyten werving speelt een belangrijke rol in de afweer tegen infecties en wanneer aangevinkt, kan bijdragen aan inflammatoire en auto-immuunziekten. Studeren leukocyten rekrutering in vivo is technisch een uitdaging, omdat cellen zijn snel, die zich binnen schepen diep in lichtverstrooiing weefsels. Tot op heden zijn de meeste intravitale imaging studies vereisen chirurgische voorbereiding op de bloedvaten en weefsel bloot te leggen. Om te voorkomen dat de weefselbeschadiging en ontsteking veroorzaakt door een operatie zelf, hier, beschrijven we een niet-invasieve single-cell imaging aanpak die kan worden gebruikt om witte bloedcellen handel in de muis voetzool en vingerkootjes te bestuderen. We bespreken de technische aspecten van onze 2P imaging voorbereiding en loopt de lezer door een typisch experiment van de eerste set tot en met uitvoering en het verzamelen van gegevens.

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

We beschrijven hier een niet-invasieve twee-foton (2P) microscopie aanpak om te studeren leukocyten homing in de muis voetzool. We bespreken de technische aspecten van onze weefsels beeldvorming voorbereiding en loopt de lezer door een typisch experiment van de eerste set tot en met uitvoering en het verzamelen van gegevens.

Niet-invasieve beeldvorming van Leukocyte Homing en migratie 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

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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 ...

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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 ...

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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 ...