前立腺細胞株は、親切にがんセンター、カリフォルニア大学デ​​ービス校で博士陵王瑜博士興-寺院カンフーによって提供されます。このプロジェクトは、NIH電気走助成4R33AI080604によってサポートされています。

1.培養前立腺細胞<ol><li>培養PRNS-1-1、5％CO 2、37℃で、10％FBSおよび抗生物質-抗真菌を補充したRPMI 1640培地中で100mm培養皿上とPNT2前立腺細胞。細胞が電気走実験のために80％の集密度に達するまで、毎日の培地をリフレッシュします。 </li></ol> 2.組立電気走チェンバース<ol><li>下部チャンバーの組み立て<ol><li> 2-プロパノールでプラスチックの電気走室を拭きます。注射器でチャンバーの底側に円形の開口部周りのマリングレードのシリコンシーラーを適用し、大規模なカバーガラス（ 図1）を取り付け。カバーガラスを押し下げると綿棒で余分な接着剤をふき取るために綿棒の裏側を使用してください。円形の開口部は、媒体と、電流が2インナーメディア貯水池の間に位置し、細胞の上に流れるようになります。 </li><li> 6ミリメートル×2を作るために小さなカバーガラスをカットダイヤモンドポイントマーカーと5ミリメートルスペーサー。 </li><li>チャンバーを裏返し。注射器および/または下部ガラス上の2の円形開口部の間の細胞接着10ミリメートル×25mmのチャネル表面を作成するための金属スパチュラでシリコン接着剤の2ストライプを適用する。 2円形の開口部との間にガラススペーサの2枚を接着。スペーサーを押し下げると綿棒および/または爪楊枝で余分な接着剤をふき取るために綿のアプリケーターの裏側を使用してください。 </li><li>シリコン接着剤が硬化するまで、24時間チャンバーを乾燥させる。接着剤から酢酸残基を除去するために蒸留水で一晩チャンバーを浸す。すぐに使用するためのチャンバを乾かしたり、清潔​​な容器に後で使用するためにチャンバーを保存する。 <img alt="図1" fo:content-width="5in" src="/files/ftp_upload/51973/51973fig1highres.jpg" width="500" /> 図1：底の電気走室の組み立て。 A）大カバーガラスを清浄室の底部に取り付けられている。B）Tガラススペーサの破片woは細胞が付着するための10ミリメートル×25 mmのチャンネルを作成するために2円形の開口部との間で接着されている。 </li></ol></li><li>電気走チャンバーに細胞を播種<ol><li>実験に必要な電気走室の必要とする数を準備します。各細胞株または処置は、単一電気走室を必要とする。 2-プロパノールとの電気走室を拭きます。滅菌PBSで3回チャンバーを洗浄し、チャンバ内の2円形の開口部との間の液体の流れを確認してください。 </li><li>無菌の細胞培養皿でチャンバを囲み、それらを15分間37℃で平衡化させる。 5分間37℃で5ミリリットル0.25％トリプシン-EDTAを使用して培養皿から前立腺細胞を剥離。 PBS中5ミリリットル、10％FBSをトリプシン-EDTAを中和する。 </li><li> 15ミリリットルチューブに細胞を移し、200×gで、37℃で5分間細胞を遠心する。上清を吸引し、培養培地1ml中に細胞を再懸濁する。 </li><li> 20μを取る計数チャンバーにロードし、細胞数を計算するために細胞溶液のリットル。培養培地で8×10 4細胞/ mlに細胞濃度を調整します。 </li><li> 37℃のインキュベーターから電気走室を取る。各チャンバーに細胞懸濁液の350μlのをシード。 </li><li>湿ったキムワイプまたは5％CO 2、37℃のインキュベーター内の湿度チャンバ内の培養皿に一晩チャンバー中で細胞をインキュベートする。 </li></ol></li></ol><img alt="図2" fo:content-width="5in" src="/files/ftp_upload/51973/51973fig2highres.jpg" width="500" /> 図2：チャンバーに細胞を播種下部チャンバはA）前立腺細胞を、トリプシン処理し、計数し、チャンバーに移し、一晩インキュベートし、乾燥し、そして洗浄されるB）トップカバーガラスを撮影する前に、チャンバをシールするために装着される。 <ol start="3"><li> A上部チャンバーをssembling <ol><li>撮影の前に上部チャンバーを組み立てます。顕微鏡は、一度に単一の電気走チャンバを撮像収容することができる。各電気走室37℃で培養培地10mlを温める。 37℃の加温プレートにインキュベーターから電気走室を転送します。 </li><li>未付着細胞を除去し、培養培地で細胞を洗浄します。チャンバ内の培地を400μlのままにしておきます。両方のガラススペーサの上に高真空グリースを適用するために注射器を使用してください。 </li><li>チャンバーをシールするために小さなカバーガラスを追加します。静かに綿棒の裏側にカバーガラスを下に押してください。綿棒と過剰メディアを拭き取ってください。 </li><li>ガラス表面を乾燥させ、カバーグラスとチャンバとの間の隙間をシールするために高真空グリースを塗布。グリースを広めるために金属スパチュラを使用してください。インナー貯水池への培地の4ミリリットルを加え、貯水池の間に液体の流れを確認してください。 </li></ol></li><li>寒天を作るブリッジ<ol><li> 2インチ長いPVC管（16分の503 ID X 5/16 OD X 1/16ウォール）のペアをカットし、反転し、100ミリリットルのビーカーにそれらを挿入します。 </li><li> 200mgのバクト寒天を測定し、2％寒天ゲルを作るために10ミリリットルの培地で50mlフラスコに追加します。 30秒間電子レンジ。転送ピペットでプラスチックチューブに寒天ゲルをロードします。寒天を固化させる10分間室温で寒天橋のままにしておきます。 </li><li>電気走チャンバの外側のリザーバへの2mlの培地を加える。電流を流すために内側と外側の媒質貯水池に寒天ブリッジを挿入します。 </li></ol></li></ol><img alt="図3" fo:content-width="3.5in" src="/files/ftp_upload/51973/51973fig3highres.jpg" width="350" /> 図3：チャンバーに細胞をロケ電気走室の最終組み立てを実証するための図を。。チャンバは完全に高真空グリースでシールされている。媒体はtで添加されるOリザーバを充填すると、2つの寒天橋がチャンバ内に挿入される。その後、電気走チャンバは顕微鏡ステージに転送され、電極は電界を印加するために取り付けられている。組み立てられた電気走チャンバの側面図は、電流が寒天ブリッジとカバーガラスとの間の空間を通ってセルの上を流れることを実証するために示されている。 3.タイムラプスイメージング<ol><li>前イメージングへの空気循環2時間、5％CO 2で37℃に環境室のスイッチをオンにします。 </li><li>顕微鏡に切り替え、画像取得ソフトウェアを開始する。顕微鏡ステージを校正し、10倍の対物レンズを選択します。 </li><li>顕微鏡ステージに電気走室を移し、テープで室を確保し、細胞に焦点を当てる。右側のカソードと外側の貯水池への電気走電極を挿入します。ワイヤーとテープで電極を固定します。 </li><li>パワーBOに切り替えxは、チャンバに電界を印加する。 25ミリメートル、長室（100 MV / mm）のための2.5ボルトに到達するために、チャンバ全体の電圧計で出力電圧を測定します。出力電流を調整することによって、実験中の電界強度を維持する。 </li><li> 10タイムラプスムービーを生成するソフトウェアでフィルムにチャンバー全体で10ポイントを選択します。 2時間10分間隔で取得条件を設定する。 </li><li>撮影を開始し、必要に応じて出力電流を調整します。 </li><li>実験の最後に、ステージからチャンバを除去し、95％アルコールで細胞を固定する。きれいにカミソリの刃で開放室を破ると、それを再使用しています。 </li></ol>電気走4.定量<ol><li>タイムラプス映画や再オリエント画像のトップにカソードを回転させます。細胞トラッキングソフトウェアに映画をエクスポートします。 </li><li>手動で各映画（ 図4のA、B）から各時点で10〜20細胞の（x、y）の位置を追跡する。南北方向の相対移動距離及び角度は、電界の同じ向きは、細胞追跡ソフトウェア（ 図4C）で計算される。 注：平均移動速度は、総移動距離と総撮影時間から変換された。方向性コサイン値に角度から変換される。細胞は、ランダムな方向性と移行する場合、ネット余弦の平均はゼロに近くなります。細胞は陰極に向かって直接移行すると、余弦値は1になります。細胞は陽極に向かって直接移動する場合は、コサイン値は-1になります。 </li></ol><img alt="図4" fo:content-width="4in" src="/files/ftp_upload/51973/51973fig4highres.jpg" width="400" /> 図4：細胞の画像を追跡線の細胞指向性を測定するために追跡A）オーバーレイ。細胞の（x、y）の位置はmanuaあるLLYタイムラプスムービーで追跡。 。細胞がランダムに移行する場合、平均余弦がゼロに近いB）しかし、細胞はカソードまたはアノードに向かって移動した場合、平均余弦の値が+（カソード）またはに近い- 。（陽極）1.0 C ）方向性は、移行の角度（θ）から変換された余弦値、によって提示されます。コサイン（θ）は、B（電界の方向に投影距離）を遠ざける距離a（移動距離）の比に等しい。 <ol start="3"><li>測定結果を保存します。組み合わせた結果を計算するために、データベース·アプリケーションにデータをインポートします。 </li><li>棒グラフ（ 図5）をプロットするためにスプレッドシートに平均移動速度と平均余弦値の結合されたデータをエクスポートします。 </li></ol>

<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 authors have nothing to disclose.

セルの電気走応答の分析は、多くの細胞の遊走又は増殖27,28を処理するための重要な機能の指標となっている。ここでは二つの前立腺細胞株をフィルムにガラス表面にカスタムメイドのチャンバーを用いる。これらの細胞株は、電気走の異なる程度を実証し、我々は、細胞内局在または電気走媒介タンパク質の活性化は、電気走応答において観察された差が生じる、不死化細胞株を生...

内因性の電界は、皮膚1、32、33、2、脳のような種々の組織で検出される。生理学的な電界が神経突起5,6の伸長を案内し、上皮および角膜創傷閉鎖1,7を促進導く胚発生3,4を含む特定の生物学的機能を提供しています。 インビトロ 、培養細胞に直流電界を印加する模倣生理電界指向性細胞遊走、または電気走を引き起こす。電気走線維芽細胞8、魚ケラチノサイト9、ヒト上皮および角膜ケラチノサイト10-12に検討されている、神経芽2、および神経前駆細胞14,13リンパ球。 （ - ）極印加磁界にさらされたときに、研究された細胞の大部分は、陰極に向かって一方向に移行する。しかし、高転移を含むいくつかの癌細胞ヒト乳癌細胞およびヒト前立腺癌細胞株PC-3Mは、陽極（+）極15、16に移動する。いくつかの機構が電気走を媒介するか、活性化を含む電界を感知する細胞の能力を説明するために提案されているEGFは、12を受容と、上皮ナトリウムチャネル17は 、PI3KおよびPTEN 18、及びカルシウムの放出メカニズムはまだ完全には理解されていない。15、19のイオンとは、複数のシグナル伝達経路が電気走に関与している可能性がある。 ここで実証する2次元電気走法が付着し、運動性細胞の指向性遊走を特徴付けるために、いずれか18、20。この技術は、から変更され、コンフルエント細胞のシートの個々の細胞遊走10、12、17または遊走をモニターするために有用である鵬とJaffe氏21、及び西村らカスタムメイド、透明なPVC室と10、取り外し可能なcoverslとIPSなどの免疫蛍光イメージングなどの二次分析用の電気走、後に簡単にセルの取得を可能にする。電気走室のガラス表面は、高倍率でかつ蛍光標識された細胞での撮影を可能にする、光互換性がある。また、そのような表面コーティングまたは電荷を変化させるように、ガラス表面の改質に実験計画を可能にする。第1カバーガラス製のスペーサーは、細胞上の電流の流れを最小限にするためにチャンバ内で使用されている。したがって、電流の流れの二乗に比例するジュール加熱は、実験中の細胞を過熱しないでしょう。接続寒天ブリッジは細胞と電極との直接接触を防止し、電気走時の培地のpHまたはイオン濃度の変化を防止する。 二つの非腫瘍形成性ヒト前立腺細胞株は、本研究での電気走応答を調べた。 PRNS-1-1 22とPNT2 23は、両方SV4です低または前立腺特異抗原（PSA）の発現なしに上皮マーカーのサイトケラチン5、8、18と19を表す0不死化、増殖因子依存性細胞株。両方の細胞株は、正常な上皮細胞の多角形の形態を維持するが、染色体異常は、染色体分析22、24で観察された。PRNS-1-1とPNT2はほとんどの実験で同様の挙動を共有するが、それらは腺房構造の形成との違いを示して行う電気走。 3-Dマトリックス、マトリゲル上で、PRNS-1-1細胞は、正常な前立腺組織25に類似したルーメンを有する中空腺房構造を形成する。しかし、PNT2細胞が管腔または偏上皮26せずに固体スフェロイドを形成する。 PRNS-1-1細胞はまた、現在の研究でPNT2よりも高いgalvanotactic応答を実証する。 PRNは、1-1腺房構造と電気走の形成の間の相関はgalvanotactic信号が広報を組織において役割を果たし得ることを示唆しているostate腺組織の内因性電界に応答して動き、そして、これら2細胞株とを区別するためにさらなる特性を提供する。

<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>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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

生理学的な電場は、胚発生、神経突起成長および上皮創傷治癒における細胞移動を導くような特定の生物学的機能を提供しています。 in vitroで培養細胞に直流電界を印加すると、方向性細胞遊走、または電気走を引き起こす。ここで実証する2次元電気走法は、カスタムメイドのポリ（塩化ビニル）（PVC）チャンバ、ガラス表面、白金電極と細胞が撮像された電動ステージを用いて修飾される。 PVC室と白金電極は、低い細胞毒性を示し、手頃な価格の、再使用可能である。ガラス表面と電動顕微鏡ステージは、画像の品質を改善し、細胞へのガラス表面や治療への可能な修飾を可能にする。我々は2つ​​の非腫瘍形成、SV40不死化前立腺細胞株、PRNS-1-1とPNT2の電気走を撮影。これら2つの細胞株は、同様の移行速度を示し、両方の側に移動カソードが、彼らは電気走における方向性の異なる程度を示します。このプロトコルを使用して得られた結果は、PRNは、1-1 PNT2細胞株は、それらの方向性遊走応答を支配する異なる固有の機能を有することができることを示唆している。

前立腺細胞（PRNS-1-1とPNT2）の2行は、この方法で調査した。両方のライン中の細胞は、2時（ 図5A）の過程で1.0 +/- 0.3ミクロン/分の同じような速度で移動する。しかし、電場の方向性はPNT2ライン（ 図5B）のためPRNS-1-1ラインの0.7 +/- 0.3、および0.2 +/- 0.8である。結果は、それらが位置手がかりに異なる指向性応答を生じる種々の細胞シグナル伝達機構を有していることを...

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.

前立腺細胞の方向性のある移動を研究するためにカスタム設計された電気走チェンバースを利用

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 ビュー.  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.

ビデオ: 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.

トカゲの運動の速度と方向の視覚感度のテスト

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

生物学

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.

細胞間の高分子輸送アッセイプランタで微粒子銃砲撃を活用

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.

タイムラプスビデオ顕微鏡を用いた細胞のランダムな移行の定量分析

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.

個々の準備ショウジョウバエ</emライブイメージングのための>エッグチェンバース

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.

間葉転換を誘導し、上皮の分析

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.

腸によって脂質輸送を研究するためのCaco-2細胞を用いました

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.

ゼブラフィッシュにおける腎毒素マイクロインジェクションは急性腎障害をモデルに

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.

細胞内局在や人身売買を監視するpHluorinタグ付き受容体を利用します

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

中間径フィラメントと微小管 2次元直接確率光再構成顕微鏡イメージング

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

A Proximal Culture Method to Study Paracrine Signaling Between Cells

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Paracrine or juxtacrine signaling mediates such interactions. The use of a conditioned medium and coculture studies are the most common methods to discriminate between these two types of interactions. However, the effect of localized high concentrations of secreted factors in the microenvironment during the paracrine interactions is not accurately recapitulated by conditioned medium and, thus, may lead to imprecise conclusions. To overcome this problem, we have devised a proximal culture method to study paracrine signaling. The two cell types are grown on either surface of a 10 &#181;m-thick polycarbonate membrane with 0.4 &#181;m pores. The pores allow the exchange of secreted factors and, at the same time, inhibit juxtacrine signaling. The cells can be collected and lysed at the endpoint to determine the effects of the paracrine signaling. In addition to allowing for localized concentration gradients of secreted factors, this method is amenable to experiments involving prolonged periods of culture, as well as the use of inhibitors. While we use this method to study the interactions between ovarian cancer cells and the mesothelial cells they encounter at the site of metastasis, it can be adapted to any two adherent cell types for researchers to study paracrine signaling in various fields, including tumor microenvironment, immunology, and development.

Paracrine and juxtacrine cellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Here, a proximal culture method is used to study paracrine signaling where the localized concentrations of the secreted factors are maintained while preventing direct cellular contact.