Name: dendra2 photoswitching through the mammary imaging window
Uploaded: 2009-06-05T00:00:00
Description: Watch this Scientific Journal Video about Dendra2 Photoswitching through the Mammary Imaging Window at JoVE.com

(; CA100324 um JC, JES und JW; U54GM064346 zu JvR CA77522 zu JES, U54CA126511 um JC und BG) Diese Arbeit wurde vom US Department of Defense (BC075554 zu BG und BC061403 zu DK), US-amerikanischen National Institutes of Health unterstützt. Wir danken D. Entenberg um Hilfe bei der Mikroskopie, M. Rottenkolber um Hilfe bei der Herstellung der Imaging-Box und J. Pollard (Albert Einstein College of Medicine) für die F4/80 Antikörper.

1. Generation von fluoreszierenden Tumoren durch Injektion von Tumorzellen in die Brustfettpolster: <ol><li> Baue eine Zelllinie, die Dendra2 Protein (als zytoplasmatischen Marker) zu 40 bis 80% Konfluenz. </li><li> Spülen Sie Geschirr mindestens 3-mal mit PBS w / o Ca 2 + oder Mg 2 +. </li><li> 3 ml Trypsin pro 10 cm Schale und bei 37 ° C, bis die meisten der Zellen und dann lösen traf das Gericht gegen eine ebene Fläche, um sie zu schütteln. </li><li> Spülen Sie alle Zellen von der Schüs...

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	<li>Farina, K. L., Wyckoff, J. B., Rivera, J., Lee, H., Segall, J. E., Condeelis, J. S., Jones, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+motility+of+tumor+cells+visualized+in+living+intact+primary+tumors+using+green+fluorescent+protein.">Cell motility of tumor cells visualized in living intact primary tumors using green fluorescent protein.</a> Cancer Res. 58, 2528-2532 (1998).</li><li>Naumov, G. N., Wilson, S. M., MacDonald, I., Schmidt, E. E., Morris, V. L., Groom, A. C., Hoffman, R. M., Chambers, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+expression+of+green+fluorescent+protein,+coupled+with+high-resolution+in+vivo+videomicroscopy,+to+monitor+steps+in+tumor+metastasis.">Cellular expression of green fluorescent protein, coupled with high-resolution in vivo videomicroscopy, to monitor steps in tumor metastasis.</a> J Cell Sci. 112, 1835-1842 (1999).</li><li>Brown, E. B. <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+measurement+of+gene+expression,+angiogenesis+and+physiological+function+in+tumors+using+multiphoton+laser+scanning+microscopy.">In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy.</a> Nature Med. 7, 864-868 (2001).</li><li>Wang, W., Wyckoff, J. B., Frohlich, V. C., Oleynikov, Y., Huttelmaier, S., Zavadil, J., Cermak, L., Bottinger, E. P., Singer, R. H., White, J. G., Segall, J. E., Condeelis, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+behavior+in+metastatic+primary+mammary+tumors+correlated+with+gene+expression+patterns+revealed+by+molecular+profiling.">Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling.</a> Cancer Res. 62, 6278-6288 (2002).</li><li>Sidani, M., Wyckoff, J., Xue, C., Segall, J. E., Condeelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+microenvironment+of+mammary+tumors+using+multiphoton+microscopy.">Probing the microenvironment of mammary tumors using multiphoton microscopy.</a> J Mammary Gland Biol Neoplasia. 11, 151-163 (2006).</li><li>Wyckoff, J. B., Wang, Y., Lin, E. Y., Li, J. F., Goswami, S., Stanley, E. R., Segall, J. E., Pollard, J. W., Condeelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+visualization+of+macrophage-assisted+tumor+cell+intravasation+in+mammary+tumors.">Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors.</a> Cancer Res. 67, 2649-2656 (2007).</li><li>Egeblad, M., Ewald, A. J., Askautrud, H. A., Truitt, M. L., Welm, B. E., Bainbridge, E., Peeters, G., Krummel, M. F., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+stromal+cell+dynamics+in+different+tumor+microenvironments+by+spinning+disk+confocal+microscopy.">Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy.</a> Dis Model Mech. 1, 155-167 (2008).</li><li>Mempel, T. R., Henrickson, S. E., von Adrian, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+priming+by+dendritic+cells+in+lymph+nodes+occurs+in+three+distinct+phases.">T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases.</a> Nature. 427, 154-159 (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 USA. 100, 2604-2609 (2003).</li><li>Kedrin, D., Gligorijevic, B., Wyckoff, J., Verkhusha, V. V., Condeelis, J., Segall, J. E., van Rheenen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+metastatic+behavior+through+a+mammary+imaging+window.">Intravital imaging of metastatic behavior through a mammary imaging window.</a> Nat. Methods. 5, 1019-1021 (2008).</li><li>Chudakov, D. M., Lukyanov, S., Lukyanov, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+intracellular+protein+movements+using+photoswitchable+fluorescent+proteins+PS-CFP2+and+Dendra2.">Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2.</a> Nat. Protocols. 2, 2024-2032 (2007).</li></ol>

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<td>PBS w/o Ca, Mg</td>
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dendra2 photoswitching through the mammary imaging window

In den letzten zehn Jahren Intravitalmikroskopie von Brusttumoren bei Mäusen und Ratten in Single-Cell-Auflösung<sup&gt; 1-4</sup&gt; Hat in wichtige Einblicke in Mechanismen des metastasierten Verhalten wie Migration, Invasion und intravasation von Tumorzellen geführt<sup&gt; 5, 6</sup&gt;, Angiogenese<sup&gt; 3</sup&gt; Und Immunzellen Antwort<sup&gt; 7-9</sup&gt;. Vor kurzem haben wir eine Technik zur Bild orthotopen Mammakarzinom berichtet über mehrere intravitales Imaging-Sitzungen in lebenden Mäusen<sup&gt; 10</sup&gt;. Zu diesem Zweck haben wir ein Mammary Imaging Window (MIW) und optimierte Bildgebung Parameter für Dendra2 entwickelt<sup&gt; 11</sup&gt; Photoschaltung und Imaging<em&gt; In vivo</em&gt;. Hier beschreiben wir das Protokoll für die Herstellung von MIW, Einsetzen der MIW auf der Spitze eines Tumors und die Abbildung der Dendra2-markierten Tumorzellen mit Hilfe eines speziell angefertigten Bildgebung ein. Dieses Protokoll kann verwendet werden, um Bild die metastatische Verhalten von Tumorzellen in verschiedenen Mikroumgebungen in Tumoren werden und ermöglicht eine langfristige Darstellung von Blutgefäßen, Tumorzellen und Wirtszellen.

Watch this Scientific Journal Video about Dendra2 Photoswitching through the Mammary Imaging Window at JoVE.com

Dendra2 Photoswitching through the Mammary Imaging Window

Intravital Photoschaltung und Verfolgung von Dendra2-markierten Tumorzellen durch die Mammary Imaging-Fenster ist eine Technik, die uns zur Abbildung der metastatischen Verhalten von Tumorzellen in ausgewählten Tumormikromilieu über einer Zeitskala von Tagen erlaubt.

Dendra2 Photoschalten durch die Mammary Imaging Fenster

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4  has resulted in important insights into ...

Research

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4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

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Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

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Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...