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

Este trabalho foi financiado pelo Departamento de Defesa dos EUA (BC075554 a BG e BC061403 para DK), National Institutes of Health EUA (U54GM064346 para JVR; CA100324 ao JC, JES e JW; CA77522 para JES, U54CA126511 ao JC e BG). Agradecemos a D. Entenberg para ajudar com microscopia, M. Rottenkolber para ajudar na fabricação de caixa de imagem e J. Pollard (Albert Einstein College of Medicine) para o anticorpo F4/80.

1. Geração de tumores fluorescentes usando a injeção de células tumorais mamárias no teclado de gordura: <ol><li> Crescer uma linhagem de células expressando Dendra2 proteínas (como um marcador citoplasmático) para 40-80% confluência. </li><li> Enxaguar pratos, pelo menos, 3 vezes com PBS w / o Ca 2 + ou Mg 2 +. </li><li> Adicionar 3 ml de tripsina por 10 centímetros prato e incubar a 37 ° C até que a maioria das células separar e em seguida, bateu o prato contra uma superfície pla...

<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. 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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>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14025</td>
<td>&nbsp;</td>
<tr>
<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>
<tr>
<td>SCID mouse</td>
<td>&nbsp;</td>
<td>NCI</td>
<td>N/A</td>
<td>&nbsp;</td>
<tr>
<td>Culture dishes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>353003</td>
<td>Custom Order</td>
</tr>
<tr>
<td>Circular coverslip 8 mm</td>
<td>&nbsp;</td>
<td>Scientific, Inc.</td>
<td>8CIR-1-FIS</td>
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dendra2 photoswitching through the mammary imaging window

Na última década, microscopia, intravital de tumores de mama em camundongos e ratos com uma única célula de resolução<sup&gt; 04/01</sup&gt; Resultou em importantes insights sobre os mecanismos de comportamento metastático, como invasão, migração e intravasation de células tumorais<sup&gt; 5, 6</sup&gt;, A angiogênese<sup&gt; 3</sup&gt; E resposta imune de células<sup&gt; 09/07</sup&gt;. Recentemente, reportaram a técnica de imagem ortotópico carcinomas mamários através de múltiplas sessões de imagem intravital em ratos vivos<sup&gt; 10</sup&gt;. Para isso, temos desenvolvido uma janela de imagem mamária (MIW) e parâmetros de imagem otimizado para Dendra2<sup&gt; 11</sup&gt; Photoswitching e imagem<em&gt; In vivo</em&gt;. Aqui, descrevemos o protocolo para a fabricação de MIW, inserção do MIW em cima de um tumor e imagem das células Dendra2 marcado tumor utilizando uma caixa de imagem personalizado construído. Este protocolo pode ser usado para a imagem do comportamento metastático das células tumorais em microambientes distintos em tumores e permite a imagem a longo prazo dos vasos sanguíneos, células tumorais e células hospedeiras.

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

Dendra2 Photoswitching through the Mammary Imaging Window

Photoswitching intravital e acompanhamento de Dendra2 marcado células tumorais através da janela de imagem mamária é uma técnica que nos permite a imagem do comportamento metastático das células tumorais em microambientes tumorais escolhido em detrimento de uma escala temporal de dias.

Dendra2 Photoswitching através da janela de imagem mamária

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