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 border="0" cellpadding="0" cellspacing="0" width="774">
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			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:117px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:323px;">Comments/Description</td>
		</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>
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			<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

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

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.

Kertenkeleler Hareket Hız ve Yön Visual Hassasiyet Test

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

Biyoloji

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.

-Cell Hücre makromoleküler Ulaştırma Testi Planta Biyolistik Bombardıman kullanımı

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.

Time-lapse video Mikroskop ile Hücrelerinin Rastgele Göç Kantitatif Analiz

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.

Bireysel hazırlanması Drosophila</emCanlı Görüntüleme için> Yumurta Chambers

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.

Mezenkimal Geçiş indüksiyon ve Epitel Analizi

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.

Bağırsak tarafından Lipit Transport Eğitim için Caco-2 hücreleri kullanarak

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.

Zebra balığı Nephrotoxin Mikroenjeksiyon Akut Böbrek Yaralanması Model

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.

pHluorin etiketli Reseptör kullanarak Subselüler Yerelleşme ve Ticareti Monitör

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

Ara filamentler ve mikrotübüller 2 boyutlu doğrudan Stokastik optik imar mikroskobu ile görüntüleme

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.