Authors thank Mr. Kotaro Okubo for the kind assistance with SEM imaging. This work was supported by the Japan Society for the Promotion of Science Grant-in-Aid for Basic Research (B) (20310069), Grant-in-Aid for Research Activity Start-up (25880021), and by research grants from the Kurata Memorial Hitachi Science and Technology Foundation and the Nippon Sheet Glass Foundation for Materials Science and Engineering.

TiO2 hazırlanması 1. cam lamel -kaplı <ol><li> Sayı bir elmas Çizici kullanarak lamelleri. Bu, her lamelleri izlemek için değil, aynı zamanda numunenin doğru tarafı yukarı bakacak emin olmak için değil, sadece yardımcı olur. İlk pirana çözelti içine daldırılarak, sonra GKD 2 O akan, lamelleri temizleyin (H 2 SO 4: H2O 2 = 4: 1). 10 dakika sonra, GKD 2 O. iyice 8 kez lamelleri durulama N2 akışı altında lamelleri kurutun. </li><li> Radyo frekans (RF) püskürtme sistemine TiO2 hedefi ayarlayın. Bir poliimid bant kullanarak püskürtme ekipmanı örnek tutucu üzerine lamelleri takın. Püskürtme hücresindeki numune tutucu yerleştirin. Basınç 2.0 × 10 ulaşana kadar odayı boşaltın -4 Pa. </li><li> Odasına Ar gazı tanıtın ve 4.0 mTor için birikim basıncını ayarlamak. Deklanşör kapalı tutarken, yavaş yavaş artış70 W RF güç </li><li> Deklanşör açın ve 120-150 nm&#39;de (Şekil 1: Adım A) bir kalınlığa sahip bir film elde etmek üzere 15 dakika için püskürtmeyle çökeltilmesi. Filmin büyüme hızı, her bir makine için elde edilmesi gerekmektedir. </li></ol> Hücre tutmayan Film ile 2. Yüzey Kaplama <ol><li> Plazma reaktörün üreticisi tarafından sağlanan yönergeleri izleyerek, O 2 plazma ile numuneyi tedavi ederek TiO2 hidrofilleştirecek. Biz 100 sccm&#39;lik O 2 akışı ile W 200 ° C&#39;de 5 dakika boyunca numune davranın. GKD 2 O numune daldırın ve yüzey süperhidrofilik olduğunu doğrulayın. İyice N2 akışı altında yüzeyi kurulayın. </li><li> 100 ml toluen 39,6 ul OTS ekleyerek 1 mM oktadesiltriklorosilandır (OTS) çözeltisi hazırlayın. Oda sıcaklığında 1 saat için çözelti içinde numune bırakın. N 2 dolgulu eldiven torbası (: Adım B Şekil 1) içinde bu adımı gerçekleştirin. </li><li> Physisorbed moleküllerini çıkartmak için, sonicate tolüen, aseton, etanol içinde örnek ve bu sırayla 5 dakika her biri için, GKD 2 O. Taze GKD 2 O örnek dört kez durulayın ve N2 akımı altında yüzeyi kurulayın. Yüzey 100-110 ° bir temas açısına sahip hidrofobik olmalıdır. </li></ol> 3. Eski Yerinde Yüzey Desenlendirme <ol><li> Laminer akış kaputu Çalışma, yüzeyde bir elmas Scriber birkaç çizik izleri çizin. Işaretleri işlenen bölgelerin kayıtlarını tutmak ve aynı zamanda odak noktası haline mikroskopları getirmekte yardımcı olur. 5 dakika boyunca% 70 etanol içinde örnek daldırarak OTS kaplı TiO 2 sterilize edin. Daha sonra sterilize edilmiş GKD 2 O. iki kez numune durulama </li><li> 35 mm çanak örnek yerleştirin ve PC12 büyüme ortamının 2 ml ekleyin (Adım 4.2). Bir CO2 kuluçka makinesi içinde 3 saat boyunca (37 ° C) inkübe edin. Bu prosedür, serum albüminler yüzeye absorbe izin amaçlanmıştır. Adsorplanan albüminler diğer prote sonraki adsorpsiyonu inhibein ve hücreleri (Şekil 1: Adım C). </li><li> Beklerken, ters floresan mikroskop kurdu. <ol><li> Ark lambasını açmak UV filtresi küp yerleştirin ve 20X objektif merceği ayarlayın. </li><li> Işık yoğunluğu I (W cm-2) kullanılarak, bir UV-gerilimi ölçü ölçmek ve benzeri gibi (J cm -2) ve d bir doz için (saniye olarak) ışınlama süresi t hesaplayın: t = d / I. Örneğin, 200 J sm bir dozda radyasyon sağlanması için -2 600 mW cm -2 arasında bir ışık kaynağı kullanılarak, ışınlama 333 sn gereklidir. </li><li> Bir sahne mikrometre kullanın ve bölgenin büyüklüğü, örneğin, 200 mikron, ışınlanmış olması ayarlamak için alan diyafram kapatın. </li></ol></li><li> (Nihai konsantrasyon 300 ug ml -1 Col-IV) 3 saat inkübasyondan sonra, 3.0 mg ml-1 tür IV kolajen 200 ul ortam ek. </li><li> Mikroskop sahneye 35 mm çanak aktarın. Karalama m bulark, örnek yüzeyi üzerine bir mikroskop odak ve 200 J cm -2 (Şekil 1: Basamak D) bir dozda UV ışığını ışın tedavisi. UV ışınlaması alanı alan diyafram açılmasını ayarlayarak ya da tahkim geometri metal maskesi alanında diyafram değiştirerek ya da değişmiş olabilir. </li><li> (Col-IV olmadan) taze büyüme ortamı ile orta değiştirin ve inkübatör geri numuneyi yerleştirin. </li></ol> 4. Hücre Kültürü <ol><li> PC12 hücrelerinin rutin kültürü kollajen kaplı plastik tabak üzerinde gerçekleştirilmektedir. <ol><li> Kat Col-IV ile 60 mm doku kültürü çanak için önce GKD 2 O ile yüzey ıslak ve tüm GKD 2 O. aspire </li><li> 300 ug ml hazırlayın -1 Col-IV GKD 2 O. ile (3 mg ml -1) 10x orijinal bir çözüm seyreltilmesi ile </li><li> 300 ug ml -1 60 mm tabak başına Col-IV, 200 ul ekleyin. Yüzeyden yayılan bir çözüm olsun. </li><li> Kuru to yaklaşık 1 saat boyunca, bir laminar akış başlığı içinde çanak. </li><li> Dulbecco fosfat tamponlu tuz (D-PBS) 4 ml ile iki kez yüzey durulayın. Hemen kaplı çanak kullanmadığınız zaman, GKD 2 O. 4 ml ile bir kez yıkayın çanak GKD 2 O aspire sonra, inkübatör çanak saklayın. Daha fazla iki hafta boyunca saklanan tutmaya çalışın. </li></ol></li><li> PC12 hücreleri oluşan bir büyüme ortamında büyütülür: Dulbecco&#39;nun modifiye edilmiş Eagle ortamı (düşük glikoz) +% 10 cenin sığır serumu +% 5 at serumu +% 1 penisilin-streptomisin solüsyonu. Bir CO2 inkübatöründe inkübe hücreleri (37 ° C,% 5 CO2), ve her gün ortamın yarısı değiştirin. Passage hücreleri izdiham ulaşmadan. <ol><li> Aspire büyüme ortamı ve ek 2 ml PBS + (D-PBS + 10 mg mi -1 sığır serum albümini (BSA), 10 mM EDTA), 37 ° C&#39;ye ön ısıtılmıştır. 37 ° C&#39;de 5 dakika süreyle inkübe edilir. Yavaşça tüm ce ayırmak için çanak dokununlls. </li><li> 15 ml konik tüp hücreleri toplayın. 37 ° C&#39;ye ön ısıtılmıştır taze D-PBS 3 ml çanak yıkayın. </li><li> 4 dakika boyunca 150 x g&#39;de santrifüj tüpü. </li><li> Süpernatanı havalandırın, ve büyüme ortamı 1 ml. </li><li> 1: 3: Rutin kültür için, hücreler 1 bölünmüş 5. </li></ol></li><li> UV-modifiye OTS / TiO2 numune üzerinde hücreleri plaka hücre yoğunluğu saymak ve 35 mm çanak (Şekil 1: Adım E) 3.0 × 10 5 hücre eklemek için. 1-2 gün boyunca nemlendirilmiş bir inkübatör çanak (37 ° C,% 5 CO2) inkübe edin. Hem saf ve sinir büyüme faktörü (NGF) -differentiated PC12 hücreleri desen deneyleri için de kullanılabilir. NGF farklılaşma için, 7S-NGF&#39;nin 100 ng ml -1 numune üzerine hücreleri kaplama önce büyüme ortamı birkaç gün eklenmektedir. NGF farklılaşma PC12 hücrelerinin adhesibility artırmak gibi görünüyor ve özellikle situ desenlendirme e, kolay taşıma yaparxperiments. </li></ol> 5. Yerinde Yüzey Desenlendirme <ol><li> Ex-situ modifiye yüzey üzerinde kültür 1-2 gün sonra hücreler ekleyerek ve UV-ışınlanmış bölgeye sadece büyüyor onaylayın. Adım 3.4 anlatıldığı gibi, mikroskop ayarlayın. </li><li> Büyüme ortamı içeren yeni bir 35 mm doku kültürü çanağı numune aktarın (NGF diferansiye hücrelerin kullanılması halinde) 100 ng ml -1 NGF ve 100 ug ml -1 Col-IV. </li><li> Mikroskop sahnede çanak yerleştirin. Uygun yerini bulur ve 200 J cm -2 (Şekil 1: Adım F, G) bir dozda UV ışığını ışın tedavisi. Yeni oluşturulan hoşgörülü bölge Şekil 1&#39;de bir yıldızla gösterilir: Adım G. </li><li> 30 dakika içinde tek bir örnek işlenmesini tamamlamaya çalışın. Işınlama sonrası, Col-IV olmadan geri ortama örnek aktarmak. </li><li> Cu ile çanak taşırken çok dikkatli olunltured hücreleri ve çanak numune transfer desenli hücrelerin ayırma engellemek için çanak. </li></ol><img alt="figür 1" src="/files/ftp_upload/53045/53045fig1highres.jpg" width="700" /> Genel sürecin 1. şematik çizimi Şekil. Ayrıntılar için metne bakınız. <a href="https://www.jove.com/files/ftp_upload/53045/53045fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

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The authors have nothing to disclose.

Mevcut protokolde, TiO2 filmi RF-manyetik alanda sıçratma tarafından kuruldu. Bize tekrarlanabilir bir alt-nm pürüzlülüğü ile fotokatalitik TiO2 filmi hazırlamak için izin verir çünkü biz birikimi bu yöntemi tercih. Püskürtülerek bırakma süreçleri malzeme bilim adamları ve mühendisleri elektronik tanıdık olmasına rağmen, biyologlar oldukça erişilebilir olmayabilir. Bu durumda, spin-kaplı TiO2 filmi alternatif bir seçenek 23 olacaktır. Bu yöntem...

Yarıiletken litografi süreçleri ve türevleri - Böyle fotolitografi 1,2, elektron ışını litografi 3-6 ve 7-10 yazdırma microcontact olarak - şimdi tanımlanmış bir konum ve geometri yaşayan hücrelerin büyümesini hücre biyolojisinde kurulmuş bir araç haline gelmiştir. Model verme yöntemi, permisif olmayan arka plan hücre permisif kaplamanın mikro ada oluşan mikrofabrike substratların kullanımına dayanır. Bu gibi alt-tabaka desen hücreleri için bir şablon olarak hizmet vermektedir. Bu teknolojiler bize hücrelerinin içsel özelliklerinin ayıklamak ve hücre bazlı ilaç tarama 11 verimini artırmak için, bir tek ve çok hücresel düzeyde hücreleri ve bunların fonksiyonlarını mühendisi yeni yöntemler sağlamıştır. Şablon desen geometrisi, yani yerinde değişmiş olabilir eğer hücreler s kültüre iken derecesi serbestlik hücre desenleme büyük ölçüde, artıracak, sert bir. Atmosferde veya vakumda örnekleri işleme sinyalinden oluşumu için olan klasik yöntemler, doğrudan, burada uygulanamamaktadır. Bu nedenle, çeşitli yeni yüzey modifikasyon teknikleri sadece birkaç isim için, fotoreaktif bileşikler 12,13 veya lazer ablasyon 5,14 tarihinde, örneğin, dayandığı öne sürülmüştür. Önerilen yöntemler güzel Choi ve ark., 16 ile ve Nakanishi&#39;nin 17 tarafından daha yakın. Robertus ark 15 gözden ve oylandı. İşte bu makalede, biz bir titanyum dioksit organik moleküllerin fotokatalitik ayrışma yararlanır in-situ yüzey modifikasyonu, (TiO2) yüzey 18,19 yeni bir protokol açıklar. Bu yöntemde, bir TiO2 filmi cam alt tabaka ve hücreleri arabirimleri, organik film arasında eklenir ve organik film yerel olarak ultraviyole (UV) ışınıma tabi tutulması işlemiyle in situ parçalanırÇevrede bir bölge (λ &lt;388 nm) ışık. Yeni protokolü hücre dışı matris proteinleri ve canlı hücreler, hem ex situ ve in situ micropatterns oluşturmak için kullanılabileceğini göstermektedir. TiO2 hücre kültürü deneylerinde tanıtmak kolaylığına özellikleri olan, biyolojik olarak uyumlu, kimyasal olarak stabil, optik olarak saydamdır. Bu protokol, hücre kültürü ortamında hücre kültürü iskeleleri değiştirmek için bir malzeme bilimi tabanlı bir alternatif sunuyor.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass coverslip</td>
			<td>Warner Instruments</td>
			<td>CS-15R15</td>
			<td>15 mm, #1.5 thickness</td>
		</tr>
		<tr>
			<td>Diamond scriber</td>
			<td>Ogura Jewel Industry</td>
			<td>D-Point Pen</td>
			<td></td>
		</tr>
		<tr>
			<td>RF sputtering system</td>
			<td>ANELVA</td>
			<td>SPC350</td>
			<td></td>
		</tr>
		<tr>
			<td>TiO2 sputtering target</td>
			<td>Kojundo Chemical Lab</td>
			<td>Titanium (IV) oxide, target</td>
			<td>Purity, 99.9%</td>
		</tr>
		<tr>
			<td>Plasma reactor</td>
			<td>Yamato</td>
			<td>PR301</td>
			<td></td>
		</tr>
		<tr>
			<td>n-octadecyltrichlorosilane 
			(OTS)</td>
			<td>Aldrich</td>
			<td>104817</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Wako</td>
			<td>204-01866</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-culture dish (35 mm)</td>
			<td>Greiner</td>
			<td>627160</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-culture dish (60 mm)</td>
			<td>BD Falcon</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>Type-IV collagen</td>
			<td>Nitta Gelatin</td>
			<td>Cellmatrix Type IV</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium (DMEM)</td>
			<td>Gibco</td>
			<td>11885-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>12483-020</td>
			<td>Heat-inactivate and pass through a 0.22 mm filter before use</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Gibco</td>
			<td>26050-088</td>
			<td>Pass through a 0.22 mm filter before use</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (100x)</td>
			<td>Nacalai tesque</td>
			<td>26253-84</td>
			<td></td>
		</tr>
		<tr>
			<td>7S nerve growth factor (NGF)</td>
			<td>Alomone Labs</td>
			<td>N-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Dojindo</td>
			<td>N001</td>
			<td>Stock solution in 0.5 M</td>
		</tr>
		<tr>
			<td>TiO2 nanoparticle</td>
			<td>Tayca</td>
			<td>TKD-701</td>
			<td></td>
		</tr>
	</tbody>
</table>

photopatterning proteins and cells in aqueous environment using tio2 photocatalysis

Titanyum dioksit yüzeyi (TiO2) üzerinde adsorbe organik kirletici ultraviyole (UV) ışık altında fotokatalizle çürümüş olabilir. Burada yerel alt tabaka yüzeyinin hücre yakınlık değiştirmek için TiO2 fotokatalizden kullanan yeni bir protokol açıklar. Bu deney için, ince bir TiO2 Film püskürtme ile kaplanmış bir cam lamel, ve TiO2 yüzey, daha sonra hücre yapışmasını inhibe eden oktadesiltriklorosilandır (OTS) türetilen bir organosilan tek tabaka ile modifiye edildi. Örnek bir hücre kültürü ortamı içine daldırılır ve UV ışığı sekizgen bölgeye ışınlandı odaklanmıştır. Bir nöronal hücre çizgisi PC12 hücreleri numune üzerine kaplandı, hücreler sadece UV-radyasyona alana bağlı kalmıştır. Biz daha hücre alt-tabaka üzerinde büyüyen bile bu yüzey modifikasyonu, aynı zamanda örneğin, in situ gerçekleştirilebilir göstermektedir. Yüzeyin düzgün modifikasyonu bir hücre dışı matris p gereklikollajen rotein UV ışınlaması sırasında ortam içinde mevcut olması mümkündür. Burada sunulan tekniğin potansiyel kültürü altında hücreleri Coculture sistemlerini inşa veya keyfi işlemek için desenleme çoklu hücre tiplerinde kullanılabilir.

Şekil 2A, püskürtme ile tevdi TiO2 filmin bir kesit tarama elektron mikroskobu (SEM) görüntüsünü göstermektedir. Gözlemlerden sonra filmin kalınlığı yaklaşık 150 nm olarak tahmin edilmiştir. Burada dikkat çekici tevdi TiO2 filmin düzlüğü olduğunu. Atomik kuvvet mikroskobu (AFM) ile daha fazla analiz yüzeyinin kök ortalama kare (rms) pürüz 0.2 nm (Şekil 2B) olduğunu ortaya koymuştur. TiO2 yüzeyi ...

Watch this Scientific Journal Video about Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis at JoVE.com

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

TiO kullanarak sulu bir ortamda Proteinler ve Hücreler photopatterning<sub&gt; 2</sub&gt; Photocatalysis

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe ...

photopatterning-proteins-cells-aqueous-environment-using-tio2

Research

JoVE Journal

Bioengineering

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

7.8K Görüntüleme.  Tohoku University. We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

Video: Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

Nükleik Asitler, Proteinler ve Küçük Moleküller Reagentless Algılama Elektrokimyasal DNA Biyosensörler Fabrikasyon

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Biyomühendislik

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology 1. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes 2, 3. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice 4, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast 5. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes 6, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.
In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.

Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these protocols easily and to utilize negative stain EM as a routine assay, in addition to other biochemical assays, for evaluating their protein samples.

Negatif Leke EM, Proteinler ve makromoleküler kompleksler görselleştirme: Izgara Hazırlık Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural ...

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

Üç boyutlu Matrix floresan etiketli Proteinler ifade Geçiş Hücreler Canlı hücre Görüntüleme

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native &beta;-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFD&alpha; and &beta;, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFD&alpha; and &beta; improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via &beta;-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

Sulu Ortamlarda Hidrokarbon Dönüşüm Enable A Toolkit

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

RootChip kullanarak Kök Çevre Rapid manipülasyonu ile Arabidopsis Kök Büyüme Time-lapse Floresans Görüntüleme

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

Sulu İki fazlı sistemler kullanma Hücre Co-kültür desenlendirme

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Kas Yenilenmesinin Fare Modellerinde İndüklenmiş Pluripotent Kök Hücre türevi Mezoangioblast benzeri Miyojenik Progenitörlerin Nakli

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

Lüminesans Rezonans Enerji memeli hücrelerinde ifade Membran Proteinleri Konformasyonel Değişiklikleri Eğitim transfer

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In conventional lab-on-a-chip-platforms, solvents and samples are passed through defined microfluidic channels with complex flow control installations. The droplet actuation platform presented here is a promising alternative. With it, it is possible to move a liquid drop (microreactor) on a planar surface of a reaction platform (lab-in-a-drop). The actuation of microreactors on the hydrophobic surface of the platform is based on the use of magnetic forces acting on the outer shell of the liquid drops which is made of a thin layer of superhydrophobic magnetite particles. The hydrophobic surface of the platform is needed to avoid any contact between the liquid core and the surface to allow a smooth movement of the microreactor. On the platform, one or more microreactors with volumes of 10 &#181;L can be positioned and moved simultaneously. The platform itself consists of a 3 x 3 matrix of electrical double coils which accommodate either neodymium or iron cores. The magnetic field gradients are automatically controlled. By variation of the magnetic field gradients, the microreactors' magnetic hydrophobic shell can be manipulated automatically to move the microreactor or open the shell reversibly. Reactions of substrates and corresponding enzymes can be initiated by merging the microreactors or bringing them into contact with surface immobilized catalysts.

Lab-in-a-drop reaction systems allow the versatile implementation of complex reactions in a microfluidic scale. An automated actuation platform consisting of a 3 x 3 matrix of electromagnetic coils was developed and successfully used to merge two 10 &#181;L microreactors and thereby initiate an enzymatic reaction in the resulting liquid marbles.

Sulu damlacıkları enzimatik Microreactors ve onların elektromanyetik çalıştırma kullanılan

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Desenlendirme Proteinlerin ve Hücreleri Çok Yönlü Yöntemi

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...