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

Ce travail a été soutenu par le Département américain de la Défense (BC075554 à BG et BC061403 à DK), US National Institutes of Health (U54GM064346 d&#39;JvR; CA100324 à JC, JES et JW; CA77522 à JES, U54CA126511 à JC et BG). Nous remercions D. Entenberg de l&#39;aide pour la microscopie, M. Rottenkolber pour les aider dans la fabrication de la boîte de l&#39;imagerie et J. Pollard (Albert Einstein College of Medicine) pour l&#39;anticorps F4/80.

1. Génération des tumeurs fluorescentes à l&#39;aide d&#39;injection de cellules tumorales mammaires dans le pavé gras: <ol><li> Cultivez une lignée cellulaire exprimant Dendra2 protéines (comme un marqueur cytoplasmique) à 40 - 80% de confluence. </li><li> Rincer la vaisselle au moins 3 fois avec du PBS w / o Ca 2 + ou Mg 2 +. </li><li> Ajouter 3 ml de trypsine par 10 cm de plat et incuber à 37 ° C jusqu&#39;à ce que la plupart des cellules se détachent et ensuite frappé du plat cont...

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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. 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		<th>Catalogue Number</th>
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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>
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<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

Dans la dernière décennie, la microscopie intravitale des tumeurs mammaires chez la souris et le rat à une seule cellule de résolution<sup&gt; 1-4</sup&gt; A abouti à des informations importantes sur les mécanismes du comportement métastatique telles que la migration, l&#39;invasion et intravasation des cellules tumorales<sup&gt; 5, 6</sup&gt;, L&#39;angiogenèse<sup&gt; 3</sup&gt; Et la réponse immunitaire des cellules<sup&gt; 7-9</sup&gt;. Nous avons récemment rapporté une technique à l&#39;image orthotopique carcinomes mammaires sur plusieurs séances d&#39;imagerie intravitale chez la souris vivante<sup&gt; 10</sup&gt;. Pour cela, nous avons développé une fenêtre d&#39;imagerie mammaire (MIW) et des paramètres optimisés pour l&#39;imagerie Dendra2<sup&gt; 11</supPhotocommutation&gt; et l&#39;imagerie<em&gt; In vivo</em&gt;. Ici, nous décrivons le protocole pour la fabrication de MIW, l&#39;insertion de la MIW sur le dessus d&#39;une tumeur et l&#39;imagerie des cellules tumorales Dendra2 marqué à l&#39;aide d&#39;une boîte d&#39;imagerie sur mesure. Ce protocole peut être utilisé pour l&#39;image du comportement métastatique des cellules tumorales dans des microenvironnements distincts dans les tumeurs et permet à l&#39;imagerie à long terme des vaisseaux sanguins, les cellules tumorales et les cellules hôtes.

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

Dendra2 Photoswitching through the Mammary Imaging Window

Photocommutation intravitale et de suivi des Dendra2 marqué les cellules tumorales à travers la fenêtre d&#39;imagerie mammaire est une technique qui permet d&#39;imager le comportement métastatique des cellules tumorales dans des microenvironnements tumoraux choisis sur une échelle de temps de jour.

Dendra2 photocommutation travers la fenêtre d&#39;imagerie mammaire

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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