prostat hücre hatları nazik Kanser Merkezi, UC Davis Dr. Ling-Yu Wang ve Dr. Hsing-Jien Kung tarafından sağlanmaktadır. Bu proje NIH galvanotaksis hibe 4R33AI080604 tarafından desteklenmektedir.

1. Kültürleme Prostat Hücreleri <ol><li> Kültür prns-1-1% 5 CO2 ile 37 ° C&#39;de% 10 FBS ve antibiyotik antimikotik ile takviye edilmiş RPMI 1640 ortamı içinde, 100 mm kültür çanakları üzerinde ve PNT2 prostat hücreleri. Hücreler galvanotaksis deneyler için% 80 confluency ulaşana kadar her gün kültür ortamı yenileyin. </li></ol> 2. Montaj galvanotaksis Odaları <ol><li> Alt odalarını Montaj <ol><li> 2-propanol ile plastik galvanotaksis odası silin. Bir şırınga ile bir bölmenin alt tarafındaki dairesel açıklıklar etrafında deniz grade silikon astarı uygulayınız ve büyük bir coverglass (Şekil 1) takın. Coverglass bastırın ve pamuklu aşırı tutkal silmek için bir pamuk aplikatör arka tarafını kullanın. Yarı dairesel açıklıklar orta ve elektrik akımı, iki iç, orta rezervuar arasında yer alan hücreler üzerinde akmasına izin verir. </li><li> 6 mm x 2 yapmak için küçük bir coverglass kesinBir elmas noktası işareti ile 5 mm tutucular. </li><li> Odasına çevirin. Bir şırınga ve / veya alt camına 2 dairesel açıklıklar arasındaki hücre bağlanması için bir 10 mm x 25 mm kanal yüzey oluşturmak için metal bir spatula ile silikon tutkal şeritleri 2 uygulanır. 2 dairesel açıklıklar arasındaki cam aralama 2 adet Tutkal. Aralayıcıları aşağı doğru itin ve pamuklu ve / veya kürdan ile aşırı tutkal silmek için pamuk aplikatör arka tarafını kullanın. </li><li> Silikon yapıştırıcı kürünü kadar 24 saat odasına kurutun. Tutkal, asetik asit kalıntılarını çıkarmak için damıtılmış su gece boyunca odası bekletin. Acil kullanım için odasına kurutun, veya temiz bir kap içinde daha sonra kullanılmak üzere odasına saklayın. <img alt="Şekil 1," fo:content-width="5in" src="/files/ftp_upload/51973/51973fig1highres.jpg" width="500" /> Şekil 1: bottom galvanotaksis odasının Meclisi. A) büyük coverglass) T, temiz odanın. B tabanına yerleştirilmiştirCam ayırıcılar adet 2 dairesel açıklıklar arasında yapıştırılmış wo hücreler eklemek için bir 10 mm x 25 mm kanal oluşturmak için. </li></ol></li><li> Galvanotaksis odalarına hücreleri Tohumlama <ol><li> Deney için gerekli galvanotaksis bölmelerin gerekli sayıda hazırlayın. Her bir hücre hattı veya tedavi tek galvanotaksis odası gerektirir. 2-propanol ile galvanotaksis odaları silin. Steril PBS ile 3 kez yıkayın odaları ve odalarına 2 dairesel açıklıklar arasındaki sıvı akışını kontrol edin. </li><li> Steril hücre kültür tabaklarında odacıkları içine alın ve bunları, 15 dakika boyunca 37 ° C&#39;de dengelenmeye gelmeye bırakın. 5 dakika için 37 ° C de, 5 ml% 0.25 tripsin-EDTA kullanılarak kültür kabı prostat hücreleri ayırmak. PBS içinde 5 ml% 10 FBS ile tripsin-EDTA nötralize. </li><li> 15 ml tüpler hücreleri aktarın ve 200 x g, 37 ° C&#39;de 5 dakika boyunca hücreler santrifüj. Süpernatan aspire ve kültür ortamı, 1 ml hücre yeniden askıya. </li><li> 20 μ alHücre eriyiği bir sayım odasına yüklemek ve hücre sayısını hesaplayın. Kültür ortamında 8 x 10 4 hücre / ml hücre konsantrasyonunu ayarlayın. </li><li> 37 ° C inkübatör dışına galvanotaksis odaları atın. Her bölmenin üzerine hücre süspansiyonu 350 ul Tohum. </li><li> Islak Kimwipe ya da% 5 CO2 ile 37 ° C de kuluçka makinesi içinde bir nem odasında kültür çanağı içinde gece boyunca odalarında hücreleri inkübe edin. </li></ol></li></ol><img alt="Şekil 2," fo:content-width="5in" src="/files/ftp_upload/51973/51973fig2highres.jpg" width="500" /> Şekil 2:. Bölmeye tohum hücreleri alt bölme kurutuldu ve temizlenir A) Prostat hücreleri tripsinize sayılır ve bölmeye aktarılır ve bir gece boyunca inkübe edilir B) üst coverglass filme önce bölmeyi kapatmak için takılır... <ol start="3"><li> BirEn iyi odaları ssembling <ol><li> Çekimlerden önce üst odasını birleştirin. Mikroskop sadece bir seferde tek bir galvanotaksis odasını görüntüleme ağırlayacak. Her galvanotaksis bölmesi için 37 ° C&#39;de kültür ortamında 10 ml kadar ısıtın. 37 ° C ısıtma plakasına kuluçka bir galvanotaksis odası aktarın. </li><li> Birleşmeyen hücrelerin çıkarılması için kültür ortamı ile hücrelerin durulayın. Bölme ortamın 400 ul bırakın. Her iki cam aralama üstünde yüksek vakum yağı uygulamak için bir şırınga kullanın. </li><li> Odasına mühür küçük bir coverglass ekleyin. Yavaşça bir pamuk aplikatör arka tarafı ile coverglass bastırın. Pamuklu aşırı orta siliniz. </li><li> Cam yüzeyini kurulayın ve coverglass ve odanın arasındaki boşluğu kapatmak için yüksek vakum gres sürün. Yağ yaymak için bir metal spatula kullanın. İç rezervuarlar için kültür ortamı 4 ml ekleyin ve rezervuar arasındaki sıvı akışını kontrol edin. </li></ol></li><li> Agar olunköprüler <ol><li> 2 inç uzunluğunda bir PVC boru donanımının bir çift kesme (503/16 çapa x 5/16 dış çaplı x 1/16 duvar), çevirme ve 100 ml&#39;lik bir beher içine yerleştirin. </li><li> 200 mg Bacto-Agar ölçün ve% 2 agar jel için 10 ml kültür ortamı ile bir 50 ml&#39;lik bir şişe içinde ilave edin. 30 saniye boyunca mikrodalga. Bir transfer pipet ile plastik boru agar jel yükleyin. Ağarı katılaştırmak için bir 10 dakika boyunca, oda sıcaklığında, agar köprüler bırakın. </li><li> Galvanotaksis odasının dış rezervuar 2 ml kültür ortamı ekleyin. Akımını yapmak için iç ve dış ortam rezervuar agar köprüler yerleştirin. </li></ol></li></ol><img alt="Şekil 3," fo:content-width="3.5in" src="/files/ftp_upload/51973/51973fig3highres.jpg" width="350" /> Şekil 3:. Odasının üzerine hücreleri filme bir diyagram galvanotaksis odasının son montajını göstermek için. odası tamamen yüksek vakum gres ile mühürlenir. Orta t ilave edilirO rezervuarları doldurun ve iki agar köprü odasına yerleştirilir. Daha sonra galvanotaksis odası mikroskop kademesine aktarılmaktadır ve elektrotların elektrik alanı uygulamak için eklenir. Monte galvanotaksis odasının yan görünüşüdür elektrik akımı agar köprü ve cam kapak arasındaki boşluk boyunca hücrelerin üzerine akar göstermek için gösterilmiştir. 3. Time-lapse Görüntüleme <ol><li> Önce görüntüleme dolaşan hava, 2 saat içinde% 5 CO2 ile 37 ° C çevre odasında açın. </li><li> Mikroskop açın ve görüntü elde yazılımı başlatmak. Mikroskop sahne kalibre ve 10X objektif seçin. </li><li> , Mikroskop sahneye galvanotaksis odaları transfer bant ile sabitleyin odasına ve hücrelerin odaklanın. Sağ taraftaki katot ile dış rezervuar için galvanotaksis elektrotlar yerleştirin. Tel ve bant ile elektrotlar sabitleyin. </li><li> Güç bo açınX odasına bir elektrik alanı uygulamak için. 25 mm uzunluğunda odasının (100 mV / mm) için 2.5 volt ulaşmak için odasına genelinde voltmetre ile çıkış gerilimini ölçün. Çıkış akımını ayarlayarak deney sırasında saha gücünü korumak. </li><li> On Time-lapse filmleri oluşturmak için yazılım film odasının karşısında 10 puan seçin. 2 saat 10 dakika aralıklarla satın alma koşulları kurun. </li><li> Filme başlatın ve gerektiği gibi çıkış akımını ayarlayın. </li><li> Deneyin sonunda, sahneden odasına çıkarın ve% 95 alkol ile hücreleri tamir. Temizlemek için bir jilet ile açık odasını kırın ve tekrar kullanın. </li></ol> Galvanotaksis 4. miktarının belirlenmesi <ol><li> Time-lapse film ve yeniden orient görüntülerin üstüne katot döndürün. Hücre izleme yazılımı filmleri verin. </li><li> El ile her bir film (Şekil 4A, B), her bir zaman noktasında 10-20 hücrelerinin (x, y) konumu etiket.kuzey-güney doğrultusunda göre göç mesafeleri ve açıları, elektrik alanının aynı yönelimi, hücre izleme yazılımı (Şekil 4C) olarak hesaplanacaktır. NOT: Ortalama göç hızı, toplam göç mesafesi ve toplam filme zaman dönüştürülür. yön kosinüs değeri açılardan dönüştürülür. Hücreler rastgele yönlülük ile geçirirseniz, net kosinüs ortalaması sıfıra yakın olacaktır. Hücreler katot doğru doğrudan geçirirseniz, kosinüs değeri +1 olacaktır. Hücreler anot doğru doğrudan geçirirseniz, kosinüs değeri -1 olur. </li></ol><img alt="Şekil 4," fo:content-width="4in" src="/files/ftp_upload/51973/51973fig4highres.jpg" width="400" /> Şekil 4: Hücre izleme yönünü ölçmek için hücre görüntüleri ile izleme hatlarının A) Kaplama.. Hücrelerin (x, y) konumu manua olanLly zaman atlamalı filmleri izlenir. . Hücreler rastgele göç varsa, ortalama kosinüs sıfıra yakın B) Ancak, hücreler katot veya anot doğru göç halinde, ortalama kosinüs değeri + (katot) ya da yakın -. (Anot) 1.0 ° C ) yön göç açıları (θ) dönüştürülür kosinüs değeri, tarafından sunulmaktadır. kosinüs (θ) mesafe b (elektrik alanının yönüne öngörülen mesafe) uzaklığa bir (göç mesafe) oranına eşittir. <ol start="3"><li> Ölçümleri kaydedin. Kombine sonuçları hesaplamak için bir veritabanı uygulaması veri almak. </li><li> Çubuk grafikler (Şekil 5) çizmek için bir tablo ortalama göç hızı ve ortalama kosinüs değerinin kombine İhracat verileri. </li></ol>

<ol>
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(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.

Bir hücrenin galvanotaksis tepki analizi pek çok hücresel göçmen veya büyüme için önemli bir fonksiyonel gösterge 27, 28 süreçleri olmuştur. Burada iki prostat hücre hatları film cam yüzeyi ile bir ısmarlama odasına kullanın. Bu hücre hatları galvanotaksis farklı derecelerde gösterdi, ve hücre içi lokalizasyonu veya galvanotaksis-sağlayan proteinlerin aktivasyonu galvanotaksis karşılık olarak gözlenen farkın neden ölümsüz hücre sıraları oluşturma işlemi sırasında müda...

Endojen elektrik alanlar, cilt 1, 32, 33 ve beyin 2 gibi çeşitli dokularda, tespit edilir. fizyolojik elektrik alanı nöron 5, 6 büyümesinin yol ve epitelyal kornea yara kapanması 1, 7 teşvik yönlendiren embriyo gelişimi 3, 4 de dahil olmak üzere, belirli bir biyolojik fonksiyonları yerine getirir,. in vitro kültürlenmiş hücrelere doğru akım elektrik alanı uygulanması taklit fizyolojik elektrik alanı ve yön hücre göçü, ya da galvanotaksis uyarmaktadır. Galvanotaksis, neuroblasts 2, ve nöronal progenitör hücreleri 14, fibroblastlar 8, balık keratinositler 9, insan epitel ve kornea keratinositler 10-12 okudu 13 lenfosit olmuştur. (-) Kutbuna uygulamalı alana maruz kaldığında, incelenen hücrelerin çoğunluğu katot doğru yönde göç. Bununla birlikte, yüksek düzeyde metastatik dahil olmak üzere çeşitli kanser hücreleri,insan göğüs kanseri hücreleri ve insan prostat kanseri hücre kuşağı PC-3M, anodal (+) kutup 15, 16 hareket eder. Çeşitli mekanizmalar galvanotaksis aracılık aktivasyonu da dahil olmak üzere elektrik alanını duymak üzere hücrelerin yeteneği, açıklamak için öne sürülen EGF 12 reseptörleri olan, epitel sodyum kanalı 17, PI3K ve PTEN 18, ve kalsiyum serbest mekanizması henüz tam olarak anlaşılamamıştır. 15, 19 iyonların ve birden çok sinyal yollarının galvanotaksis katılan mümkündür. Burada ortaya 2 boyutlu galvanotaksis yöntemi yapışık, hareketli hücrelerin belli bir yönde geçişini tanımlamak için, ya da 18, 20. Bu teknik, değiştirilmiş olan birleşik hücre tabakasının bireysel hücre göçü 10, 12, 17 ya da göç izlemek için yararlıdır Peng ve Jaffe 21, ve Nishimura ve ark. ısmarlama, açık PVC odaları ile 10, çıkarılabilir coversl ileIPS gibi bağışıklık floresan görüntüleme gibi ikincil analizi için galvanotaksis, sonra kolayca hücre alımı için izin. galvanotaksis odaları cam yüzeyi yüksek büyütmede ve floresan etiketli hücreleri ile filme sağlayan, optik-uyumludur. Aynı zamanda, bu tür bir yüzey kaplaması veya masraf değiştirilmesi gibi, cam yüzeyin modifikasyonu ile deney tasarımı sağlar. No: 1 coverglass yapılmış Aralayıcılar hücreleri üzerinde mevcut akışını en aza indirmek için odalar içinde kullanılır; Bu nedenle akım karesiyle orantılıdır joule ısıtma, deney sırasında hücreler aşırı ısınmasına olmaz. bağlayan köprüler Agar hücreleri ile elektrotların direkt temasını önlemek ve galvanotaksis sırasında ortam pH veya iyon konsantrasyonunun değişimi önlemek. İki olmayan insan prostat kanser oluşturucu hücre çizgileri bu çalışmada kullanılan galvanotaksis yanıtı için incelenmiştir. prns-1-1 22 ve PNT2 23 hem SV4 olanBüyüme faktörüne bağlı hücre çizgileri, prostat spesifik antijeni (PSA) düşük ya da hiç bir ifade ile 5, 8, 18 ve 19 sitokeratin epitel işaretlerini ifade 0-ölümsüzleştirilmiş. Her iki hücre dizisi normal epitel hücreleri çokgen morfolojisini muhafaza ama kromozom anormalliği karar verildi.İşlemi 22, 24 gözlendi. Prns-1-1 ve PNT2 çoğu deneyde içindeki davranışları paylaşmalarına rağmen bunlar asinar yapısının oluşumu ve farklılıkları gösteriyor galvanotaksis. 3-boyutlu matrisi üzerinde Matrigel, prns-1-1 hücreleri, normal prostat bezi dokusu 25 benzeyen lümen içi boş asinar yapılar oluştururlar. Bununla birlikte, PNT2 hücreleri lümen veya polarize epitel 26 olmadan katı küreler oluşturma. prns-1-1 hücreler de bu çalışmada PNT2 daha yüksek bir galvanotactic tepki göstermektedir. Listesi asinar yapısının oluşumu ve galvanotaksis arasındaki korelasyon prns-1-1 galvanotactic sinyalleri pr organize edilmesinde rol oynayabileceğini öneriyorostate bezi dokusu endojen elektrik alanlarına tepki olarak hareket ve bu 2 hücre hatları arasında ayrım yapmak için daha fazla özelliklerini içerir.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Cells&lt;/strong&gt;</td></tr><tr><td>pRNS-1-1 prostate cells</td><td></td><td></td><td>Lee, &lt;em&gt;et al.&lt;/em&gt; (1994)</td></tr><tr><td>PNT2 prostate cells</td><td>Sigma-Aldrich</td><td>95012613-1VL</td><td>Berthon, &lt;em&gt;et al.&lt;/em&gt; (1995)</td></tr><tr><td colspan="4">&lt;strong&gt;Medium and solutions&lt;/strong&gt;</td></tr><tr><td>RPMI 1640 medium&amp;nbsp;</td><td>Invitrogen</td><td>11875-093</td><td>warm up to 37 &amp;deg;C before use</td></tr><tr><td>Fetal Bovine Serum - Premium</td><td>Atlanta Biologicals</td><td>S11150</td><td>10% in PBS, warm up to 37 &amp;deg;C before use</td></tr><tr><td>Antibiotic-Antimycotic (100x)</td><td>Life Technologies</td><td>15240</td><td>add 5 ml to 500 ml medium</td></tr><tr><td>2-propanol</td><td>VWR</td><td>BDH1133-5GL</td><td></td></tr><tr><td>PBS</td><td>-</td><td>-</td><td>137 mM NaCl, 2.7 mM KCl, 4.3 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; and 1.5 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; in 1,000 ml of H&lt;sub&gt;2&lt;/sub&gt;O, pH to 7.4 and autoclaved, warm up to 37 &amp;deg;C before use</td></tr><tr><td>0.25% Trypsin-EDTA&amp;nbsp;</td><td>Invitrogen</td><td>25200-056</td><td>warm up to 37 &amp;deg;C before use, treat cells for 3-5 min at 37 &amp;deg;C</td></tr><tr><td colspan="4">&lt;strong&gt;Galvanotaxis device&lt;/strong&gt;</td></tr><tr><td>Galvanotaxis chambers</td><td>Precision Plastics Inc, CA</td><td></td><td>Custom-designed (1/4&quot; x 2&quot; x 3.5&quot;), non-toxic, clear PVC chambers. Please contact the authors for the design specs.</td></tr><tr><td>Galvanotaxis electrodes</td><td>UCD electric shop</td><td></td><td>platinum coiled electrodes with flexable cords</td></tr><tr><td>Galvanotaxis power box</td><td>Substrate Engineering, CA</td><td></td><td>custom-designed DC power output with voltmeter</td></tr><tr><td>Microscope Cover Glass, Large, 45 x 50 mm, No. 1.5</td><td>Fisher</td><td>12-544-F</td><td></td></tr><tr><td>Microscope Cover Glass, small, 25 x 25 mm, No. 1</td><td>ThermoScientific</td><td>3307</td><td></td></tr><tr><td>Diamond point marker</td><td>ThermoScientific</td><td>750</td><td></td></tr><tr><td>Marine grade silicon sealer, clear</td><td>3M</td><td>051135-08019</td><td></td></tr><tr><td>High vacuum grease</td><td>Dow Corning</td><td>2021846-0807</td><td></td></tr><tr><td>6 ml syringe</td><td>Fisher Scientific</td><td>05-561-64</td><td></td></tr><tr><td>Nichiryo Syringe, 1.5 ml</td><td>Nichiryo</td><td>SG-M</td><td></td></tr><tr><td>Cotton applicators</td><td>Purtian Medical Products</td><td>806-WC</td><td></td></tr><tr><td>Qtips</td><td>Johnson &amp;amp; Johnson</td><td>729389</td><td></td></tr><tr><td>Nalgene 180 PVC tubing&amp;nbsp;</td><td>Nalgene</td><td>8000-9030</td><td>503/16 ID x 5/16 OD x 1/16 Wall</td></tr><tr><td>Bacto-Agar</td><td>Difco</td><td>0140-01</td><td>make 2% agar solution</td></tr><tr><td>Razor Blade</td><td>Personna</td><td>74-0001</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipments and Software&lt;/strong&gt;</td></tr><tr><td>Benchtop Centrifuge</td><td>Eppendorf</td><td>5810R</td><td>operated with an A-4-62 rotor</td></tr><tr><td>Cellometer Auto T4</td><td>Nexcelom</td><td>Auto T4</td><td></td></tr><tr><td>Cellometer counting chambers</td><td>Nexcelom</td><td>CHT4-SD100-002</td><td>load 20 &amp;#956;l cell solutions to count</td></tr><tr><td>Culture Temp Warming plate</td><td>Bel-Art Scienceware</td><td>370150000</td><td>to keep the galvanotaxis chambers at 37 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Eclipse TE-2000 microscope with motorized stage and environmental chamber</td><td>Nikon</td><td></td><td></td></tr><tr><td>Plan Fluor 10X/0.30 objective len</td><td>Nikon</td><td></td><td></td></tr><tr><td>Retiga EX CCD camera&amp;nbsp;</td><td>Qimaging</td><td></td><td>Cooled CCD camara, mono-color, 12-bit</td></tr><tr><td>Compressed air with 5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Airgas</td><td></td><td>special order</td></tr><tr><td>Volocity 6.3</td><td>PerkinElmer</td><td></td><td>Image acquiring software</td></tr><tr><td>Improvision OpenLab 5.5.2</td><td>PerkinElmer</td><td></td><td>Cell tracking software and customized to measure migration angles</td></tr><tr><td>FileMaker Pro Advanced, 8.0</td><td>FileMaker</td><td></td><td></td></tr><tr><td>Microsoft Excel 2008 for Mac</td><td>Microsoft</td><td></td><td></td></tr></tbody></table>

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

fizyolojik elektrik alan gibi embriyo gelişimi, nöronal akıbet ve epitel yara iyileşmesinde hücre göçü yönetmenlik gibi belirli biyolojik fonksiyonları, hizmet vermektedir. In vitro olarak kültürlenen hücrelere doğru akım elektrik alanı uygulanması yönlü hücre göçü veya galvanotaksis neden olur. Burada ortaya 2 boyutlu galvanotaksis yöntemi ölçüye poli (vinil klorür) (PVC) odaları, cam yüzeyi, platin elektrot ve hücreler, görüntülü edildiği, bir motorlu kaide kullanımı ile modifiye edilir. PVC odaları ve platin elektrotları düşük sitotoksisite gösterirler ve uygun fiyatlı ve yeniden kullanılabilir vardır. Cam yüzeyi ve motorlu mikroskop sahne görüntülerin kalitesini artırmak ve hücrelere cam yüzeyi ve tedavileri mümkün değişikliklere izin. Biz olmayan iki tümörijenik, SV40-ölümsüzleştirdi prostat hücre hatları, prns-1-1 ve PNT2 ve galvanotaksis filme. Bu iki hücre hatları benzer göç hızları göstermek ve hem de doğru göçkatot, ancak galvanotaksis yönelmişlik farklı bir derecede gösteriyor. Bu protokol ile elde edilen sonuçlar prns-1-1 ve PNT2 hücre hatları kendi yönlü göç yanıtları yöneten farklı içsel özelliklere sahip olabileceğini düşündürmektedir.

Prostat hücreleri (prns-1-1 ve PNT2) iki satır, bu yöntemle araştırılmıştır. Her iki hatta hücreler 2 saate (Şekil 5A) boyunca 1.0 +/- 0.3 mikron / dakika arasında benzer hızlarda göç ederler. Bununla birlikte, elektrik alanına yön prns-1-1 hattı için 0.7 +/- 0.3 ve PNT2 hattı (Şekil 5B) 0.2 +/- 0.8. Sonuçlar, pozisyonel ipuçları farklı yön yanıtlarına neden farklı hücresel sinyal mekanizmalarına sahip olduğunu düşündüren, (100 hücre izlendi p &lt;0....

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.

Prostat Hücrelerinin Yönlü Göç Eğitim için özel tasarlanmış galvanotaksis Chambers kullanılması

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 Görüntüleme.  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

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

2D ve 3D Ortamlarda Sinir Progenitör Hücreleri Elektrik Alanı kontrollü Yönetmen Göç

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Biyomühendislik

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

Bölümlere Mikroakışkan Cihazlar ve Canlı Hücre Görüntüleme kullanma Kanser Kök Hücre Göç Değerlendirilmesi

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

Tıp

Migratory Behavior of Cells Generated in Ganglionic Eminence Cultures

Migration of cells is a common process that leads to the development and maturation of the vertebrate central nervous system (Hatten, '99). The cerebral cortex consists of two basic neuronal types: excitatory and inhibitory. These cells arise in distinct areas and migrate into the cortex along different routes (Pearlman et al., '98). Inhibitory interneurons migrate tangentially from subcortical sources, mostly from different regions of the ganglionic eminences (Gelman et al., '09; Xu et al., '04). Their movement requires precise spatiotemporal control imposed by environmental cues, to allow for the establishment of proper cytoarchitecture and connectivity in the cerebral cortex (Caviness &amp; Rakic, '78; Hatten, '90; Rakic, '90). To study the migratory behavior of cells generated in proliferative zones of the ganglionic eminences (GE) in newborn ferrets in vitro we used a 3 dimensional culture arrangement in a BD Matrigel Matrix. The culture setup consisted of two GE explants and a source of tested proteins extracted from the cerebral cortex and adsorbed on fluorescent latex Retrobeads IX positioned between the explants (Hasling et al., '03; Riddle et al., '97). After 2-3 days of culture, the cells start to appear at the edge of the explant showing a propensity to leave the tissue in a radial direction. Live imaging allowed observation of migratory patterns without the necessity of labeling or marking the cells. When exposed to fractions of the protein extract obtained from isochronic ferret cortex, the GE cells displayed different behaviors as judged by quantitative kinetic analysis of individual moving cells.

Time lapse imaging of 3D tissue culture allows studying migratory behavior of individual cells originating from ganglionic eminence in reaction to fractionated protein extract from cerebral cortex.

Gangliyonik Eminence Kültürler oluşturuldu Hücre Göçmen Davranış

Migration of cells is a common process that leads to the development and maturation of the vertebrate central nervous system (Hatten, '99). The cerebral ...

Nörobilim

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Canlı hücre görüntüleme deneyleri için çalışma Glioblastoma Beyin tümör kök hücre göç ve işgali

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Kanser Araştırmaları

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

Bir Modifiye Zigmond Odası Testi kullanılarak Trunk Sinir Crest Hücre Göç Analizi

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

Microfabricated Kanallar Hücre Göç Çalışması

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

Dıştan Uygulanan Doğru Akım Elektrik Alanında Nöral Prekürsör Hücre Göç Kinetik Analizi için galvanotaksis Assay

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

Planar Gradient Difüzyon Sistemi 3D Kollajen Matrix Kemotaksis Araştırma

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

ECIS / Taksiler ile Hücresel Kemotaksis Ölçümü

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

Biyoloji

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Görüntüleme G Nötrofil gibi HL60 Hücreleri Kemotaksis ve Sinyal Olaylar Reseptör aracılı Protein-çiftli

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...