The prostate cell lines are kindly provided by Dr. Ling-Yu Wang and Dr. Hsing-Jien Kung at Cancer Center, UC Davis. This project is supported by NIH galvanotaxis grant 4R33AI080604.

1. Culturing Prostate Cells
<ol>
	<li>Culture the pRNS-1-1 and PNT2 prostate cells on 100 mm culture dishes in RPMI 1640 medium supplemented with 10% FBS and Antibiotic-Antimycotic at 37 &#176;C with 5% CO2. Refresh the culture medium every day until the cells reach 80% confluency for the galvanotaxis experiments.</li>
</ol>
2. Assembling Galvanotaxis Chambers
<ol>
	<li>Assembling bottom chambers
	<ol>
		<li>Wipe a plastic galvanotaxis chamber with 2-propanol. Apply the marine grade silicon sealer around the circular openings on the bottom side of a chamber with a syringe and attach a large coverglass (Figure 1). Use the back side of a cotton applicator to push down the coverglass and wipe off the excess glue with cotton swabs. The circular openings allow the medium and electric current to flow over the cells located between the two inner medium reservoirs.</li>
		<li>Cut a small coverglass to make 6 mm x 25 mm spacers with a diamond point marker.</li>
		<li>Flip the chamber. Apply 2 stripes of the silicon glue with a syringe and/or a metal spatula to create a 10 mm x 25 mm channel surface for cell attachment between the 2 circular openings on the bottom glass. Glue 2 pieces of glass spacer between the 2 circular openings. Use the back side of the cotton applicators to push down the spacers and wipe off the excess glue with cotton swabs and/or toothpicks.</li>
		<li>Dry the chamber for 24 hr until the silicon glue is cured. Soak the chamber in distilled water overnight to remove the acetic acid residue from the glue. Dry the chamber for immediate use, or store the chamber for later use in a clean container. 
		<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51973/51973fig1highres.jpg" width="500" /> 
	Figure 1: Assembly of the bottom galvanotaxis chamber. A) A large coverglass is attached to the bottom of a clean chamber. B) Two pieces of glass spacers are glued between the 2 circular openings to create a 10 mm x 25 mm channel for cells to attach.</li>
	</ol>
	</li>
	<li>Seeding cells on the galvanotaxis chambers
	<ol>
		<li>Prepare the require number of galvanotaxis chambers needed for the experiment. Each cell line or treatment requires a single galvanotaxis chamber. Wipe the galvanotaxis chambers with 2-propanol. Wash the chambers with sterile PBS 3 times and check the liquid flow between the 2 circular openings in the chambers.</li>
		<li>Enclose the chambers in sterile cell culture dishes and allow them to equilibrate at 37 &#176;C for 15 min. Detach the prostate cells from their culture dish using 5 ml 0.25% Trypsin-EDTA at 37 &#176;C for 5 min. Neutralize the trypsin-EDTA with 5 ml 10% FBS in PBS.</li>
		<li>Transfer the cells to 15 ml tubes and centrifuge the cells for 5 min at 200 x g, 37 &#176;C. Aspirate the supernatant and re-suspend the cells in 1 ml of culture medium.</li>
		<li>Take 20 &#181;l of cell solution to load to a counting chamber and calculate the cell number. Adjust the cell concentration to 8 x 104 cell/ml with culture medium.</li>
		<li>Take the galvanotaxis chambers out of the 37 &#176;C incubator. Seed 350 &#181;l of the cell suspension onto each chamber.</li>
		<li>Incubate the cells in the chambers overnight in the culture dish with wet Kimwipe or in a humidity chamber in the incubator at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
</ol>
	<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51973/51973fig2highres.jpg" width="500" /> 
	Figure 2: Seeding the cells to the chamber. The bottom chamber is dried and cleaned. A) Prostate cells are trypsinized, counted and transferred to the chamber and incubated overnight. B) A top coverglass is attached to seal the chamber before filming.
<ol start="3">
	<li>Assembling the top chambers
	<ol>
		<li>Assemble the top chamber prior to filming. The microscope can only accommodate imaging a single galvanotaxis chamber at one time. Warm up 10 ml of culture medium at 37 &#176;C for each galvanotaxis chamber. Transfer a galvanotaxis chamber from the incubators to a 37 &#176;C warming plate.</li>
		<li>Rinse the cells with culture medium to remove the unattached cells. Leave 400 &#181;l of medium in the chamber. Use a syringe to apply high vacuum grease on top of both glass spacers.</li>
		<li>Add a small coverglass to seal the chamber. Gently press down the coverglass with the back side of a cotton applicator. Wipe off the excess medium with cotton swabs.</li>
		<li>Dry the glass surface and apply high vacuum grease to seal the gap between the coverglass and the chamber. Use a metal spatula to spread the grease. Add 4 ml of the culture medium to the inner reservoirs and check the liquid flow between the reservoirs.</li>
	</ol>
	</li>
	<li>Make agar bridges
	<ol>
		<li>Cut a pair of 2 inch long PVC tubing (503/16 ID x 5/16 OD x 1/16 Wall), flip and insert them into a 100 ml beaker.</li>
		<li>Measure 200 mg Bacto-Agar and add it in a 50 ml flask with 10 ml culture medium to make 2% agar gel. Microwave for 30 sec. Load the agar gel to the plastic tubing with a transfer pipet. Leave the agar bridges at room temperature for 10 min to solidify the agar.</li>
		<li>Add 2 ml culture medium to the outer reservoirs of the galvanotaxis chamber. Insert the agar bridges to the inner and outer medium reservoirs to conduct the current.</li>
	</ol>
	</li>
</ol>
	<img alt="Figure 3" fo:content-width="3.5in" src="/files/ftp_upload/51973/51973fig3highres.jpg" width="350" /> 
	Figure 3: Filming the cells on the chamber. A diagram to demonstrate the final assembly of the galvanotaxis chamber. The chamber is completely sealed with high vacuum grease. Medium is added to fill the reservoirs, and two agar bridges are inserted into the chamber. Then the galvanotaxis chamber is transferred to the microscope stage and the electrodes are attached to apply the electric field. Side-view of the assembled galvanotaxis chamber is shown to demonstrate that the electric current flows over the cells through the agar bridges and the space between the cover glass.
3. Time-lapse Imaging
<ol>
	<li>Switch on the environmental chamber to 37 &#176;C with 5% CO2 in the circulating air 2 hr prior to imaging.</li>
	<li>Switch on the microscope and initiate the image-acquiring software. Calibrate the microscope stage and select the 10X objective.</li>
	<li>Transfer the galvanotaxis chambers to the microscope stage, secure the chamber with tape and focus on the cells. Insert the galvanotaxis electrodes to the outer reservoirs with cathode on the right side. Secure the wire and the electrodes with tape.</li>
	<li>Switch on the power box to apply an electric field to the chamber. Measure the output voltage with the voltmeter across the chamber to reach 2.5 volts for the 25 mm long chamber (100 mV/mm). Maintain the field strength during the experiment by adjusting the output current.</li>
	<li>Select 10 points across the chamber to film in the software to generate ten time-lapse movies. Set up the acquisition conditions at 10-min intervals for 2 hr.</li>
	<li>Start filming and adjust the output current as needed.</li>
	<li>At the end of the experiment, remove the chamber from the stage and fix the cells with 95% alcohol. Break open the chamber with a razor blade to clean and re-use it.</li>
</ol>
4. Quantification of Galvanotaxis
<ol>
	<li>Rotate the time-lapse movies and re-orient the cathode to the top of the images. Export the movies to the cell tracking software.</li>
	<li>Manually track the (x,y) position of 10-20 cells at each time-point from each movie (Figure 4 A, B). The migration distances and the angles relative to the north-south direction, the same orientation of the electric field, will be calculated in the cell tracking software (Figure 4C). 
	NOTE: The average migration speed is converted from the total migration distance and total filming time. The directionality is converted from the angles to the cosine value. If cells migrate with random directionality, the average of net cosine will be close to zero. If the cells migrate directly toward the cathode, the cosine value will be +1. If the cells migrate directly toward the anode, the cosine value will be -1.</li>
</ol>
<img alt="Figure 4" fo:content-width="4in" src="/files/ftp_upload/51973/51973fig4highres.jpg" width="400" /> 
Figure 4: Cell tracking to measure directionality. A) Overlay of the tracking lines with cell images. The (x, y) positions of the cells are manually tracked in the time-lapse movies. If the cells migrate randomly, the average cosine is close to zero. B) However, if the cells migrate toward the cathode or the anode, the value of the average cosine is close to + (cathode) or &#8211; (anode) 1.0. C) The directionality is presented by the cosine value, which is converted from the migration angles (&#952;). The cosine (&#952;) equals the ratio of the distance a (the migration distance) to distance b (the distance projected to the direction of the electric field).
<ol start="3">
	<li>Save the measurements. Import the data to a database application to calculate the combined results.</li>
	<li>Export the combined data of average migration speed and average cosine value to a spreadsheet to plot the bar graphs (Figure 5).</li>
</ol>

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The authors have nothing to disclose.

The analysis of a cell&#8217;s galvanotaxis response has been an important functional indicator for many cellular migratory or growth processes 27, 28. Here we use a custom-made chamber with glass surface to film two prostate cell lines. These cell lines demonstrated different degrees of galvanotaxis, and we speculate that the intracellular localization or the activation of the galvanotaxis-mediating proteins might be interfered during the process of generating the immortal cell lines, resulting in the observe...

Endogenous electric fields are detected in various tissues, such as skin 1, 32, 33 and brain 2. The physiological electric field serves specific biological functions, including directing embryo development 3, 4, guiding the outgrowth of neuronal processes 5, 6 and promoting epithelial and corneal wound closure 1, 7. In vitro, application of a direct current electric field to cultured cells mimics the physiological electric field and induces directional cell migration, or galvanotaxis. Galvanotaxis has been studied in fibroblasts 8, fish keratinocytes 9, human epithelial and corneal keratinocytes 10-12, lymphocytes 13, neuroblasts 2, and neuronal progenitor cells 14. When exposed to the applied field, the majority of studied cells migrate directionally toward the cathodal (-) pole. Yet, several cancer cells, including highly metastatic human breast cancer cells and the human prostate cancer cell line PC-3M, move to the anodal (+) pole 15, 16. Several mechanisms are proposed to mediate galvanotaxis or to explain the ability of the cells to sense the electric field, including activation of EGF receptors 12, the epithelial sodium channel 17, PI3K and PTEN 18, and release of calcium ions 15, 19. The mechanism is not yet fully understood and it is possible that multiple signaling pathways are involved in galvanotaxis.
The 2-dimensional galvanotaxis method we demonstrate here is useful to characterize the directional migration of adherent, motile cells, either to monitor individual cell migration 10, 12, 17 or migration of a sheet of confluent cells 18, 20. This technique is modified from Peng and Jaffe21, and Nishimura et al.10 with custom-made, clear PVC chambers, with removable coverslips allowing for easy cell retrieval after galvanotaxis for secondary analysis, such as immuno-fluorescence imaging. The glass surface of the galvanotaxis chambers is optical-compatible, which allows the filming at high magnification and with fluorescently-labeled cells. It also allows the experimental design with modification of the glass surface, such as changing the surface coating or charges. Spacers made of No. 1 coverglass are used in the chambers to minimize the current flow over the cells; therefore the joule heating, which is proportional to the square of the current flow, would not overheat the cells during the experiment. The connecting agar bridges prevent direct contact of the electrodes with the cells and prevent change of the medium pH or ion concentration during galvanotaxis.
Two non-tumorigenic human prostate cell lines were examined for their galvanotaxis response in this study. The pRNS-1-1 22 and PNT2 23 are both SV40-immortalized, growth factor-dependent cell lines expressing the epithelial markers cytokeratin 5, 8, 18 and 19 with low or no expression of the prostate specific-antigen (PSA). Both cell lines maintain the polygonal morphology of normal epithelial cells, but chromosome abnormality was observed in karyotyping 22, 24. Although pRNS-1-1 and PNT2 share similar behaviors in most experiments, they do show differences in the formation of acinar structure and in galvanotaxis. On a 3-D matrix, Matrigel, the pRNS-1-1 cells form hollow acinar structures with lumens resembling the normal prostate gland tissue 25. However, the PNT2 cells form solid spheroids without a lumen or polarized epithelium 26. The pRNS-1-1 cells also demonstrate a higher galvanotactic response than the PNT2 in the current study. The correlation between the formation of acinar structure and galvanotaxis in pRNS-1-1 suggests that the galvanotactic signals may play a role in organizing the prostate gland tissue movements in response to endogenous electric fields, and provides further characteristics to discriminate between these 2 cell lines.

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			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:117px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:323px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:216px;">Cells</td>
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			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;">pRNS-1-1 prostate cells</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td>Lee et. al., (1994)</td>
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			<td height="21" style="height:21px;">PNT2 prostate cells</td>
			<td style="width:117px;">Sigma-Aldrich</td>
			<td style="width:119px;">95012613-1VL</td>
			<td>Berthon et al., (1995)</td>
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			<td height="21" style="height:21px;">Medium and solutions</td>
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			<td height="21" style="height:21px;">RPMI 1640 medium&#160;</td>
			<td style="width:117px;">Invitrogen</td>
			<td style="width:119px;">11875-093</td>
			<td>warm up to 37&#176;C before use</td>
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			<td height="42" style="height:42px;width:216px;">Fetal Bovine Serum - Premium</td>
			<td style="width:117px;">Atlanta Biologicals</td>
			<td style="width:119px;">S11150</td>
			<td>10% in PBS, warm up to 37&#176;C before use</td>
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		<tr height="42">
			<td height="42" style="height:42px;">Antibiotic-Antimycotic (100x)</td>
			<td style="width:117px;">Life Technologies</td>
			<td style="width:119px;">15240</td>
			<td>add 5mL to 500mL medium</td>
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			<td height="21" style="height:21px;width:216px;">2-propanol</td>
			<td style="width:117px;">VWR</td>
			<td>BDH1133-5GL</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:117px;">-</td>
			<td style="width:119px;">-</td>
			<td>137mM NaCl, 2.7mM KCl, 4.3mM Na2HPO4 and 1.5mM KH2PO4 in 1000mL of H2O, pH to 7.4 and autoclaved, warm up to 37&#176;C before use</td>
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			<td height="21" style="height:21px;">0.25% Trypsin-EDTA&#160;</td>
			<td style="width:117px;">Invitrogen</td>
			<td style="width:119px;">25200-056</td>
			<td>warm up to 37&#176;C before use, treat cells for 3-5 min at 37&#176;C</td>
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			<td height="21" style="height:21px;">Galvanotaxis device</td>
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			<td height="42" style="height:42px;width:216px;">Galvanotaxis chambers</td>
			<td style="width:117px;">Precision Plastics Inc, CA</td>
			<td style="width:119px;"></td>
			<td>custom-designed (1/4&#34; X 2&#34; X 3.5&#34;), non-toxic, clear PVC chambers</td>
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			<td height="42" style="height:42px;width:216px;">Galvanotaxis electrodes</td>
			<td style="width:117px;">UCD electric shop</td>
			<td style="width:119px;"></td>
			<td>platinum coiled electrodes with flexable cords</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Galvanotaxis power box</td>
			<td style="width:117px;">Substrate Engineering, CA</td>
			<td style="width:119px;"></td>
			<td>custom-designed DC power output with voltmeter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microscope Cover Glass, Large, 45X50mm-No. 1.5</td>
			<td style="width:117px;">Fisher</td>
			<td style="width:119px;">12-544-F</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microscope Cover Glass, small, 25X25mm, No. 1</td>
			<td style="width:117px;">ThermoScientific</td>
			<td style="width:119px;">3307</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Diamond point marker</td>
			<td style="width:117px;">ThermoScientific</td>
			<td style="width:119px;">750</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Marine grade silicon sealer, clear</td>
			<td style="width:117px;">3M</td>
			<td style="width:119px;">051135-08019</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">High vacuum grease</td>
			<td style="width:117px;">Dow Corning</td>
			<td style="width:119px;">2021846-0807</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6mL syringe</td>
			<td style="width:117px;">Fisher Scientific</td>
			<td style="width:119px;">05-561-64</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nichiryo Syringe, 1.5mL</td>
			<td style="width:117px;">Nichiryo</td>
			<td style="width:119px;">SG-M</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cotton applicators</td>
			<td style="width:117px;">Purtian Medical Products</td>
			<td style="width:119px;">806-WC</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Qtips</td>
			<td style="width:117px;">Johnson &#38; Johnson</td>
			<td style="width:119px;">729389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nalgene 180 PVC tubing&#160;</td>
			<td style="width:117px;">Nalgene</td>
			<td style="width:119px;">8000-9030</td>
			<td>503/16 ID x 5/16 OD x 1/16 Wall</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Bacto-Agar</td>
			<td style="width:117px;">Difco</td>
			<td style="width:119px;">0140-01</td>
			<td>make 2% agar solution</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Razor Blade</td>
			<td style="width:117px;">Personna</td>
			<td style="width:119px;">74-0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Equipments and Software</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Benchtop Centrifuge</td>
			<td style="width:117px;">Eppendorf</td>
			<td style="width:119px;">5810R</td>
			<td>operated with an A-4-62 rotor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellometer Auto T4</td>
			<td style="width:117px;">Nexcelom</td>
			<td style="width:119px;">Auto T4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cellometer counting chambers</td>
			<td style="width:117px;">Nexcelom</td>
			<td style="width:119px;">CHT4-SD100-002</td>
			<td>load 20uL cell solutions to count</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Culture Temp Warming plate</td>
			<td style="width:117px;">Bel-Art Scienceware</td>
			<td style="width:119px;">370150000</td>
			<td>to keep the galvanotaxis chambers at 37&#176;C&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Eclipse TE-2000 microscope with motorized stage and environmental chamber</td>
			<td style="width:117px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plan Fluor 10X/0.30 objective len</td>
			<td style="width:117px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Retiga EX CCD camera&#160;</td>
			<td style="width:117px;">Qimaging</td>
			<td style="width:119px;"></td>
			<td>Cooled CCD camara, mono-color, 12-bit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Compressed air with 5% CO2&#160;</td>
			<td style="width:117px;">Airgas</td>
			<td style="width:119px;"></td>
			<td>special order</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Volocity 6.3</td>
			<td style="width:117px;">PerkinElmer</td>
			<td style="width:119px;"></td>
			<td>Image acquiring software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Improvision OpenLab 5.5.2</td>
			<td style="width:117px;">PerkinElmer</td>
			<td style="width:119px;"></td>
			<td>Cell tracking software and customized to measure migration angles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FileMaker Pro Advanced, 8.0</td>
			<td style="width:117px;">FileMaker</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microsoft Excel 2008 for Mac</td>
			<td style="width:117px;">Microsoft</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

utilizing custom-designed galvanotaxis chambers to study directional migration of prostate cells

The physiological electric field serves specific biological functions, such as directing cell migration in embryo development, neuronal outgrowth and epithelial wound healing. Applying a direct current electric field to cultured cells in vitro induces directional cell migration, or galvanotaxis. The 2-dimensional galvanotaxis method we demonstrate here is modified with custom-made poly(vinyl chloride) (PVC) chambers, glass surface, platinum electrodes and the use of a motorized stage on which the cells are imaged. The PVC chambers and platinum electrodes exhibit low cytotoxicity and are affordable and re-useable. The glass surface and the motorized microscope stage improve quality of images and allow possible modifications to the glass surface and treatments to the cells. We filmed the galvanotaxis of two non-tumorigenic, SV40-immortalized prostate cell lines, pRNS-1-1 and PNT2. These two cell lines show similar migration speeds and both migrate toward the cathode, but they do show a different degree of directionality in galvanotaxis. The results obtained via this protocol suggest that the pRNS-1-1 and the PNT2 cell lines may have different intrinsic features that govern their directional migratory responses.

Two lines of prostate cells (pRNS-1-1 and PNT2) were investigated with this method. Cells in both lines migrate at similar speeds of 1.0 +/- 0.3 microns/min over the course of 2-hours (Figure 5A). However, the directionality to the electric field is 0.7 +/- 0.3 for the pRNS-1-1 line, and 0.2 +/- 0.8 for the PNT2 line (Figure 5B). The results show a significant difference in the galvanotaxis of these two cell lines (p&#60;0.01, 100 cells were tracked), suggesting that they have different ...

Watch this Scientific Journal Video about Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells at JoVE.com

Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells

We present a method to apply a physiological electric field to migrating, immortalized prostate cells in a custom-made galvanotaxis chamber. Using this method, we demonstrate that 2 lines of non-tumorigenic prostate cells demonstrate different degrees of migration directionality in the field.

The physiological electric field serves specific biological functions, such as directing cell migration in embryo development, neuronal outgrowth and ...

utilizing-custom-designed-galvanotaxis-chambers-to-study-directional

Research

JoVE Journal

Biology

9.1K vistas.  University of California, Davis. We present a method to apply a physiological electric field to migrating, immortalized prostate cells in a custom-made galvanotaxis chamber. Using this method, we demonstrate that 2 lines of non-tumorigenic prostate cells demonstrate different degrees of migration directionality in the field.

Video: Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a fluorescently tagged-protein of interest is transiently expressed in plant tissue following biolistic delivery of its encoding DNA construct. The intra- and intercellular distribution of the tagged protein is then analyzed by confocal microscopy. We describe this technology in detail, providing step-by-step protocols to assay and evaluate the extent of symplastic protein transport in three plant species, Arabidopsis thaliana, Nicotiana benthamiana and N. tabacum (tobacco).

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells