Name: utilizing custom-designed galvanotaxis chambers to study directional migration of prostate cells
Uploaded: 2014-12-07T14:00:00
Description: Watch this Scientific Journal Video about Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells at JoVE.com

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...

<ol>
	<li>Reid, B., Nuccitelli, R., Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+measurement+of+bioelectric+currents+with+a+vibrating+probe.">Non-invasive measurement of bioelectric currents with a vibrating probe.</a> Nat Protoc. 2 (3), 661-669 (2007).</li><li>Cao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+electric+currents+might+guide+rostral+migration+of+neuroblasts.">Endogenous electric currents might guide rostral migration of neuroblasts.</a> EMBO Rep. 14 (2), 184-190 (2013).</li><li>Hotary, K. B., Robinson, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+electrical+currents+and+voltage+gradients+in+Xenopus+embryos+and+the+consequences+of+their+disruption.">Endogenous electrical currents and voltage gradients in Xenopus embryos and the consequences of their disruption.</a> Dev Biol. 166 (2), 789-800 (1994).</li><li>Hotary, K. B., Robinson, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+a+role+for+endogenous+electrical+fields+in+chick+embryo+development.">Evidence of a role for endogenous electrical fields in chick embryo development.</a> Development. 114 (4), 985-996 (1992).</li><li>Yamashita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric+axon+guidance+in+embryonic+retina:+galvanotropism+revisited.">Electric axon guidance in embryonic retina: galvanotropism revisited.</a> Biochem Biophys Res Commun. 431 (2), 280-283 (2013).</li><li>Wood, M. D., Willits, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applied+electric+field+enhances+DRG+neurite+growth:+influence+of+stimulation+media,+surface+coating+and+growth+supplements.">Applied electric field enhances DRG neurite growth: influence of stimulation media, surface coating and growth supplements.</a> J Neural Eng. 6 (4), 046003 (2009).</li><li>Kucerova, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+electrical+signals+in+murine+corneal+wound+re-epithelialization.">The role of electrical signals in murine corneal wound re-epithelialization.</a> J Cell Physiol. 226 (6), 1544-1553 (2011).</li><li>Sillman, A. L., Quang, D. M., Farboud, B., Fang, K. S., Nuccitelli, R., Isseroff, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Dermal+fibroblasts+do+not+exhibit+directional+migration+on+collagen+I+in+direct-current+electric+fields+of+physiological+strength.">Human Dermal fibroblasts do not exhibit directional migration on collagen I in direct-current electric fields of physiological strength.</a> Exp Dermatol. 12 (4), 396-402 (2003).</li><li>Allen, G. M., Mogilner, A., Theriot, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoresis+of+cellular+membrane+components+creates+the+directional+cue+guiding+keratocyte+galvanotaxis.">Electrophoresis of cellular membrane components creates the directional cue guiding keratocyte galvanotaxis.</a> Curr Biol. 23 (7), 560-568 (2013).</li><li>Nishimura, K. Y., Isseroff, R. R., Nuccitelli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+keratinocytes+migrate+to+the+negative+pole+in+direct+current+electric+fields+comparable+to+those+measured+in+mammalian+wounds.">Human keratinocytes migrate to the negative pole in direct current electric fields comparable to those measured in mammalian wounds.</a> J Cell Sci. 109 (1), 199-207 (1996).</li><li>Farboud, B., Nuccitelli, R., Schwab, I. R., Isseroff, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DC+electric+fields+induce+rapid+directional+migration+in+cultured+human+corneal+epithelial+cells.">DC electric fields induce rapid directional migration in cultured human corneal epithelial cells.</a> Exp Eye Res. 70 (5), 667-673 (2000).</li><li>Fang, K. S., Ionides, E., Oster, G., Nuccitelli, R., Isseroff, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+receptor+relocalization+and+kinase+activity+are+necessary+for+directional+migration+of+keratinocytes+in+DC+electric+fields.">Epidermal growth factor receptor relocalization and kinase activity are necessary for directional migration of keratinocytes in DC electric fields.</a> J Cell Sci. 112 (12), 1967-1978 (1999).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+T+lymphocytes+migrate+toward+the+cathode+of+DC+electric+fields+in+microfluidic+devices.">Activated T lymphocytes migrate toward the cathode of DC electric fields in microfluidic devices.</a> Lab Chip. 11 (7), 1298-1304 (2011).</li><li>Meng, X., Arocena, M., Penninger, J., Gage, F. H., Zhao, M., Song, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+mediated+electrotaxis+of+embryonic+and+adult+neural+progenitor+cells+in+the+presence+of+growth+factors.">PI3K mediated electrotaxis of embryonic and adult neural progenitor cells in the presence of growth factors.</a> Exp Neurol. 227 (1), 210-217 (2011).</li><li>Wu, D., Ma, X., Lin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DC+Electric+Fields+Direct+Breast+Cancer+Cell+Migration,+Induce+EGFR+Polarization,+and+Increase+the+Intracellular+Level+of+Calcium+Ions.+.">DC Electric Fields Direct Breast Cancer Cell Migration, Induce EGFR Polarization, and Increase the Intracellular Level of Calcium Ions. .</a> Cell Biochem Biophys. 67 (3), 1115-1125 (2013).</li><li>Martin-Granados, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+PP1/NIPP1+in+steering+migration+of+human+cancer+cells.">A role for PP1/NIPP1 in steering migration of human cancer cells.</a> PLoS One. 7 (7), 40769 (2012).</li><li>Yang, H. Y., Charles, R. P., Hummler, E., Baines, D. L., Isseroff, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epithelial+sodium+channel+mediates+the+directionality+of+galvanotaxis+in+human+keratinocytes.">The epithelial sodium channel mediates the directionality of galvanotaxis in human keratinocytes.</a> J Cell Sci. 126 (9), 1942-1951 (2013).</li><li>Zhao, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+signals+control+wound+healing+through+phosphatidylinositol-3-OH+kinase-gamma+and+PTEN.">Electrical signals control wound healing through phosphatidylinositol-3-OH kinase-gamma and PTEN.</a> Nature. 442 (7101), 457-460 (2006).</li><li>Shanley, L. J., Walczysko, P., Bain, M., MacEwan, D. J., Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influx+of+extracellular+Ca2++is+necessary+for+electrotaxis+in+Dictyostelium.">Influx of extracellular Ca2+ is necessary for electrotaxis in Dictyostelium.</a> J Cell Sci. 119 (22), 4741-4748 (2006).</li><li>Zhao, M., Agius-Fernandez, A., Forrester, J. V., McCaig, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+migration+of+corneal+epithelial+sheets+in+physiological+electric+fields.">Directed migration of corneal epithelial sheets in physiological electric fields.</a> Invest Ophthalmol Vis Sci. 37 (13), 2548-2558 (1996).</li><li>Peng, H. B., Jaffe, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarization+of+fucoid+eggs+by+steady+electrical+fields.">Polarization of fucoid eggs by steady electrical fields.</a> Dev Biol. 53 (2), 277-284 (1976).</li><li>Lee, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+adult+human+prostatic+epithelial-cells+immortalized+by+polybrene-induced+DNA+transfection+with+a+plasmid+containing+an+origin-defective+sv40-genome.">Characterization of adult human prostatic epithelial-cells immortalized by polybrene-induced DNA transfection with a plasmid containing an origin-defective sv40-genome.</a> Int J Oncol. 4 (4), 821-830 (1994).</li><li>Berthon, P., Cussenot, O., Hopwood, L., Leduc, A., Maitland, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+expression+of+sv40+in+normal+human+prostatic+epithelial+and+fibroblastic+cells+-+differentiation+pattern+of+non-tumorigenic+cell-lines.">Functional expression of sv40 in normal human prostatic epithelial and fibroblastic cells - differentiation pattern of non-tumorigenic cell-lines.</a> Int J Oncol. 6 (2), 333-343 (1995).</li><li>Aurich-Costa, J., Vannier, A., Gr&#233;goire, E., Nowak, F., Cherif, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IPM-FISH,+a+new+M-FISH+approach+using+IRS-PCR+painting+probes:+application+to+the+analysis+of+seven+human+prostate+cell+lines.">IPM-FISH, a new M-FISH approach using IRS-PCR painting probes: application to the analysis of seven human prostate cell lines.</a> Genes Chromosomes Cancer. 30 (2), 143-160 (2001).</li><li>Tyson, D. R., Inokuchi, J., Tsunoda, T., Lau, A., Ornstein, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+requirements+of+prostatic+epithelial+cell+lines+for+acinar+morphogenesis+and+lumen+formation+in+vitro:+role+of+extracellular+calcium.">Culture requirements of prostatic epithelial cell lines for acinar morphogenesis and lumen formation in vitro: role of extracellular calcium.</a> Prostate. 67 (15), 1601-1613 (2007).</li><li>Lang, S. H., Sharrard, R. M., Stark, M., Villette, J. M., Maitland, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostate+epithelial+cell+lines+form+spheroids+with+evidence+of+glandular+differentiation+in+three-dimensional+Matrigel+cultures.">Prostate epithelial cell lines form spheroids with evidence of glandular differentiation in three-dimensional Matrigel cultures.</a> Br J Cancer. 85 (4), 590-599 (2001).</li><li>Babona-Pilipos, R., Popovic, M. R., Morshead, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+galvanotaxis+assay+for+analysis+of+neural+precursor+cell+migration+kinetics+in+an+externally+applied+direct+current+electric+field.">A galvanotaxis assay for analysis of neural precursor cell migration kinetics in an externally applied direct current electric field.</a> J Vis Exp. (68), (2012).</li><li>Meng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric+field-controlled+directed+migration+of+neural+progenitor+cells+in+2D+and+3D+environments.">Electric field-controlled directed migration of neural progenitor cells in 2D and 3D environments.</a> J Vis Exp. (60), (2012).</li><li>Pullar, C. E., Isseroff, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP+mediates+keratinocyte+directional+migration+in+an+electric+field.">Cyclic AMP mediates keratinocyte directional migration in an electric field.</a> J Cell Sci. 118 (9), 2023-2034 (2005).</li><li>Sheridan, D. M., Isseroff, R. R., Nuccitelli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imposition+of+a+physiologic+DC+electric+field+alters+the+migratory+response+of+human+keratinocytes+on+extracellular+matrix+molecules.">Imposition of a physiologic DC electric field alters the migratory response of human keratinocytes on extracellular matrix molecules.</a> J Invest Dermatol. 106 (4), 642-646 (1996).</li><li>Feng, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guided+migration+of+neural+stem+cells+derived+from+human+embryonic+stem+cells+by+an+electric+field.">Guided migration of neural stem cells derived from human embryonic stem cells by an electric field.</a> Stem Cells. 30 (2), 349-355 (2012).</li><li>Mukerjee, E. V., Isseroff, R. R., Nuccitelli, R., Collins, S. D., Smith, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microneedle+array+for+measuring+wound+generated+electric+fields.">Microneedle array for measuring wound generated electric fields.</a> Conf Proc IEEE Eng Med Biol Soc. 1, 4326-4328 (2006).</li><li>Nuccitelli, R., Nuccitelli, P., Li, C., Narsing, S., Pariser, D. M., Lui, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electric+field+near+human+skin+wounds+declines+with+age+and+provides+a+noninvasive+indicator+of+wound+healing.">The electric field near human skin wounds declines with age and provides a noninvasive indicator of wound healing.</a> Wound Rep. and Reg. 19, 645-655 (2011).</li></ol>

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.

<table border="0" cellpadding="0" cellspacing="0" width="774">
	<tbody>
		<tr height="42">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cells</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Medium and solutions</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-propanol</td>
			<td style="width:117px;">VWR</td>
			<td>BDH1133-5GL</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Galvanotaxis device</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
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			<td height="21" style="height:21px;">High vacuum grease</td>
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		</tr>
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			<td height="21" style="height:21px;width:216px;">6mL syringe</td>
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			<td style="width:119px;">SG-M</td>
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		</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>
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		</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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto-Agar</td>
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			<td>make 2% agar solution</td>
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			<td height="21" style="height:21px;width:216px;">Razor Blade</td>
			<td style="width:117px;">Personna</td>
			<td style="width:119px;">74-0001</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Equipments and Software</td>
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			<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>
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			<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>
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		<tr height="21">
			<td height="21" style="height:21px;">Compressed air with 5% CO2&#160;</td>
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			<td>special order</td>
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		<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 ...

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