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

Detta arbete stöddes av US Department of Defense (BC075554 till BG och BC061403 till DK), US National Institutes of Health (U54GM064346 till JvR, CA100324 till JC, JES och JW, CA77522 till JES, U54CA126511 till JC och BG). Vi tackar D. Entenberg för hjälp med mikroskopi, M. Rottenkolber om hjälp i tillverkningen av avbildning rutan och J. Pollard (Albert Einstein College of Medicine) för F4/80 antikroppen.

1. Generering av fluorescerande tumörer med hjälp av injektion av tumörceller i juverfett pad: <ol><li> Odla en cellinje som uttrycker Dendra2 protein (som cytoplasma markör) till 40 - 80% confluency. </li><li> Skölj rätter minst 3 gånger med PBS w / o Ca 2 + eller Mg 2 +. </li><li> Tillsätt 3 ml trypsin per 10 cm parabolantenn och inkubera vid 37 ° C tills de flesta celler lossnar och då slå skålen mot en plan yta att skaka den. </li><li> Skölj alla celler bort skålen, och använda...

<ol>
	<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>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14190-144</td>
<td>&nbsp;</td>
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<td>Trypsin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25300</td>
<td>&nbsp;</td>
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<td>HBSS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14025</td>
<td>&nbsp;</td>
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<td>DMEM media (MDA-MB-231 cells)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11965</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane(Aerrane)</td>
<td>&nbsp;</td>
<td>Baxter</td>
<td># NDC 10019-773-40</td>
<td>&nbsp;</td>
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<td>SCID mouse</td>
<td>&nbsp;</td>
<td>NCI</td>
<td>N/A</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>BD Falcon</td>
<td>353003</td>
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<td>Circular coverslip 8 mm</td>
<td>&nbsp;</td>
<td>Scientific, Inc.</td>
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dendra2 photoswitching through the mammary imaging window

Under det senaste decenniet, intravital mikroskopi av bröst-tumörer hos möss och råttor vid encelliga upplösning<sup&gt; 1-4</sup&gt; Har resulterat i viktiga insikter i mekanismer av metastaserande beteende som migration, invasion och intravasation av tumörceller<sup&gt; 5, 6</sup&gt;, Angiogenes<sup&gt; 3</sup&gt; Och immunceller svar<sup&gt; 7-9</sup&gt;. Vi har nyligen rapporterat en teknik för att bilden orthotopic bröst carcinom över flera intravital avbildning sessioner i levande möss<sup&gt; 10</sup&gt;. För detta har vi utvecklat ett bröst Imaging Fönster (MIW) och optimerade parametrar avbildning för Dendra2<sup&gt; 11</sup&gt; Photoswitching och bildbehandling<em&gt; In vivo</em&gt;. Här beskriver vi protokollet för tillverkning av MIW, införande av MIW på toppen av en tumör och avbildning av Dendra2-märkta tumörceller med hjälp av en specialbyggd avbildning rutan. Detta protokoll kan användas för att bilden av metastaserande beteendet hos tumörceller i olika mikromiljöer i tumörer och möjliggör långsiktiga avbildning av blodkärl, tumörceller och celler värd.

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

Dendra2 Photoswitching through the Mammary Imaging Window

Intravital photoswitching och spårning av Dendra2 märkt tumörceller via mammary Imaging Fönster är en teknik som tillåter oss att bilden av metastaserande beteendet hos tumörceller i utvalda tumör mikromiljöer över en tidsskala av dagar.

Dendra2 Photoswitching genom bröst Imaging Fönster

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

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