Name: live-cell imaging of migrating cells expressing fluorescently-tagged proteins in a three-dimensional matrix
Uploaded: 2011-12-22T13:00:00
Description: Watch this Scientific Journal Video about Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix at JoVE.com

We thank Dr. Grant Sumida for critical reading of the manuscript. This work was supported by a Beckman Young Investigator Award (SY), a Hellman Family New Faculty Award (SY), a NIH EUREKA, the University of California Cancer Research Coordinating Committee.

1. Generation of stable cell line (e.g. MDCK cells)
<ol>
	<li>Plate cells at 80-90% confluency in a p35 dish. Do not let cells form 100% confluent monolayer, which will reduce transfection efficiency.</li>
	<li>Transfect the cells with the plasmid of interest using Lipofectamine 2000. Optimize transfection conditions using manufacturer's protocol.</li>
	<li>Next day, passage the cells into two p150 petri dishes. The large dish is recommended to allow enough spacing between the stable colonies.</li>
	<li>Next day, ...

<ol>
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Here we describe a method for using live-cell imaging to study the mechanisms of cell migration in a three-dimensional matrix. The success of this technique depends on obtaining "good" clones stably expressing GFP-tagged proteins. The low level of GFP proteins will require an excess excitation exposure that compromises cell health, while too high GFP level will have undesirable side effects on the cell. Thus, the vector choice to transfect genes into cells is important (e.g., the promoter, fluorescence tag, etc). There a...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Collagen, bovine, Type I</td>
 <td>BD Biosciences</td>
 <td>354231</td>
 <td>Stock is about 3 mg/ml</td>
 </tr>
 <tr>
 <td>3-aminopropyltrimethoxysilane</td>
 <td>Sigma Aldrich</td>
 <td>281778</td>
 <td>Dilute in water</td>
 </tr>
 <tr>
 <td>glutaraldehyde</td>
 <td>Sigma Aldrich</td>
 <td>340855</td>
 <td>Dilute in PBS</td>
 </tr>
 <tr>
 <td>&nbsp;1M Hepes</td>
 <td>Invitrogen</td>
 <td>15630-080</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fluospheres polystyrene microspheres 1 &micro;m, red fluorescence (580/605)</td>
 <td>Invitrogen</td>
 <td>F13083</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Geneticin (G418)</td>
 <td>Invitrogen</td>
 <td>11811-031</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="4">Culture media components:</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Invitrogen</td>
 <td>31600-034</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum</td>
 <td>Atlanta Biologicals</td>
 <td>S115500</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin/Streptomycin</td>
 <td>Invitrogen</td>
 <td>15140-122</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Kanamycin</td>
 <td>Invitrogen</td>
 <td>15160-054</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

live-cell imaging of migrating cells expressing fluorescently-tagged proteins in a three-dimensional matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Watch this Scientific Journal Video about Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix at JoVE.com

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

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