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.

1. Preparazione di TiO2 Rivestiti vetro Coverslip <ol><li> Numero i coprioggetti usando una punta per tracciare diamante. Questo non solo aiuta a tenere traccia di ogni coprioggetto ma anche per garantire che il lato corretto del campione è rivolta verso l&#39;alto. Pulire i coprioggetti, prima sotto un getto DDH 2 O, poi immergendole in una soluzione piranha (H 2 SO 4: H 2 O 2 = 4: 1). Dopo 10 minuti, risciacquare accuratamente i coprioggetti, 8 volte in DDH 2 O. Asciugare il coprioggetto sotto N 2 flusso. </li><li> Impostare TiO 2 destinazione nel sistema sputtering a radiofrequenza (RF). Fissare i coprioggetti su un supporto campione del materiale sputtering utilizzando un nastro poliimmide. Collocare il supporto del campione nella camera di sputtering. Evacuare la camera fino a quando la pressione raggiunge 2.0 × 10 -4 Pa. </li><li> Introdurre gas Ar nella camera e impostare la pressione di deposizione a 4,0 mTorr. Pur mantenendo l&#39;otturatore chiuso, aumentare gradualmentela potenza RF a 70 W. </li><li> Aprire l&#39;otturatore e sputter per 15 minuti per ottenere una pellicola con uno spessore di 120-150 nm (Figura 1: Fase A). Tasso di crescita del film deve essere derivato per ciascuna macchina. </li></ol> 2. Surface Coating con cellulare repellente Film <ol><li> Hydrophilize superficie TiO 2 trattando il campione con O 2 al plasma, seguendo le istruzioni fornite dal produttore del reattore al plasma. Trattiamo il campione per 5 minuti a 200 W con O 2 flusso di 100 sccm. Immergere il campione in DDH 2 O e confermare che la superficie è superidrofilia. Asciugare accuratamente la superficie sotto N 2 flusso. </li><li> Preparare la soluzione 1 octadecyltrichlorosilane mm (OTS) con l&#39;aggiunta di 39,6 OTS microlitri per 100 ml di toluene. Immergere il campione nella soluzione per 1 ora a RT. Condurre questo passaggio all&#39;interno di un sacchetto 2 guanto -filled N (Figura 1: Fase B). </li><li> Per rimuovere molecole physisorbed, sonicate il campione in toluene, acetone, etanolo, e DDH 2 O per 5 minuti ciascuno, in questo ordine. Sciacquare il campione quattro volte in acqua dolce DDH 2 O e asciugare la superficie sotto N 2 flusso. La superficie deve essere idrofobica con un angolo di contatto di 100-110 °. </li></ol> 3. Ex-Situ Patterning Surface <ol><li> Lavorare in una cappa a flusso laminare, disegnare diversi segni di graffi con una punta per tracciare diamante sulla superficie. I segni aiutano a tenere traccia delle regioni elaborati e anche portare i microscopi a fuoco. Sterilizzare il TiO OTS rivestite 2 immergendo il campione in etanolo al 70% per 5 min. Risciacquare il campione due volte nel sterile DDH 2 O. </li><li> Trasferire il campione in un piatto da 35 mm, e aggiungere 2 ml di terreno di coltura PC12 (vedi punto 4.2). Incubare per oltre 3 ore in un incubatore CO 2 (37 ° C). Questa procedura è destinata a far albumine sieriche assorbono sulla superficie. Albumine adsorbite inibiscono conseguente assorbimento di altri proteins e cellule (Figura 1: fase c). </li><li> Durante l&#39;attesa, impostare il microscopio a fluorescenza invertito. <ol><li> Accendere la lampada ad arco, inserire il cubo di filtro UV, e impostare la lente dell&#39;obiettivo a 20X. </li><li> Misurare l&#39;intensità della luce I (W cm -2) usando un misuratore UV intensità, e calcolare il tempo di irradiazione t (in secondi) per una dose di d (in cm -2 J) come: t = d / I. Ad esempio, per irradiare alla dose di 200 J cm -2 utilizzando una sorgente luminosa di 600 mW cm -2, è richiesta 333 sec di irradiazione. </li><li> Utilizzare un micrometro e chiudere il diaframma di campo per impostare la dimensione della regione da irradiare, ad esempio, 200 micron. </li></ol></li><li> Dopo l&#39;incubazione 3 ore, integrare la media con 200 ml di 3,0 mg ml -1 di collagene di tipo-IV (Col-IV; finali concentrazione di 300 ug ml -1). </li><li> Trasferire il piatto 35 mm al palco microscopio. Trova il graffio marca fuoco il microscopio sulla superficie del campione, e irradiare luce UV con una dose di 200 J cm -2 (Figura 1: Fase D). La zona di irradiazione UV può essere modificata sia regolando l&#39;apertura del diaframma di campo o sostituendo il diaframma di campo con una maschera metallica della geometria arbitrale. </li><li> Sostituire la media con un mezzo di coltura fresco (senza Col-IV), e posizionare il campione torna in incubatrice. </li></ol> Cultura 4. cellulare <ol><li> Routine coltura di cellule PC12 è effettuata su un piatto di plastica rivestita di collagene. <ol><li> Per rivestire un piatto di tessuto-cultura di 60 mm, con Col-IV, prima bagnare la superficie con DDH 2 O e aspirare tutto DDH 2 O. </li><li> Preparare 300 mg ml -1 Col-IV diluendo la soluzione originale (3 mg ml -1) 10x con DDH 2 O. </li><li> Aggiungere 200 ml di 300 mcg ml -1 Col-IV per 60-mm piatto. Lasciate che la soluzione di diffondersi attraverso la superficie. </li><li> Dry tha piatto in una cappa a flusso laminare per circa 1 ora. </li><li> Lavare la superficie due volte con 4 ml di tampone fosfato salino di Dulbecco (D-PBS). Quando non si usa subito il piatto rivestito, lavare il piatto una volta con 4 ml di DDH 2 O. Dopo aspirazione DDH 2 O, riporre il piatto in incubatrice. Cercate di non conserverà per più di due settimane. </li></ol></li><li> Cellule PC12 sono coltivate in un terreno di coltura costituito da: medio (glucosio basso) modificato Eagle Dulbecco + 10% siero fetale bovino + 5% soluzione cavallo penicillina streptomicina siero + 1%. Incubare le cellule in un incubatore CO 2 (37 ° C, 5% CO 2), e sostituire la metà del mezzo ogni altro giorno. Passage cellule prima che raggiungano confluenza. <ol><li> Aspirare il terreno di coltura, e aggiungere 2 ml di PBS + (D-PBS + 10 mg ml -1 albumina di siero bovino (BSA) + EDTA 10 mM) preriscaldata a 37 ° C. Incubare per 5 min a 37 ° C. Battere delicatamente il piatto di staccare tutti cells. </li><li> Raccogliere le cellule in un tubo da 15 ml. Sciacquare il piatto con 3 ml di fresco D-PBS, preriscaldata a 37 ° C. </li><li> Centrifugare la provetta a 150 g per 4 min. </li><li> Aspirare il surnatante e aggiungere 1 ml di mezzo di crescita. </li><li> Per la cultura di routine, dividere le celle 1: 3 a 1: 5. </li></ol></li><li> Per piastra cellule sul campione modificato UV OTS / TiO 2, contare densità cellulare e aggiungere 3,0 x 10 5 cellule in un piatto da 35 mm (Figura 1: Stadio E). Incubare il piatto in incubatrice umidificata (37 ° C, 5% CO 2) per 1-2 giorni. Entrambi fattore di crescita ingenuo e nervoso (NGF), le cellule PC12 -differentiated possono essere utilizzati per gli esperimenti patterning. Per NGF differenziazione, 100 ng ml -1 di 7S-NGF viene aggiunto alla crescita media diversi giorni prima placcatura le cellule sul campione. NGF-differenziazione sembra aumentare la adhesibility delle cellule PC12 e facilita la manipolazione, specialmente nella in-situ e patterningxperiments. </li></ol> 5. In-Situ Patterning Surface <ol><li> Dopo 1-2 giorni di coltura sulla superficie ex situ modificato, confermano che le cellule sono Collegamento e cresce solo sulla regione UV irradiata. Impostare il microscopio, come descritto al punto 3.4. </li><li> Trasferire il campione in un piatto di coltura di tessuti da 35 mm contenente il mezzo di crescita, 100 ng ml -1 NGF (nel caso di utilizzo di cellule NGF dissociati), e 100 mg ml -1 Col-IV. </li><li> Porre la capsula sul palco microscopio. Trovare la posizione appropriata, e irradiare luce UV con una dose di 200 J cm -2 (Figura 1: Fase F, G). La regione permissiva appena creato viene indicato con un asterisco nella figura 1: Punto G. </li><li> Prova a completare l&#39;elaborazione di un singolo campione entro 30 minuti. Dopo l&#39;irraggiamento, trasferire il campione di nuovo nel mezzo senza Col-IV. </li><li> Fare molta attenzione quando si trasporta il piatto con cucellule ltured e trasferimento del campione dal piatto per piatto per evitare il distacco delle cellule fantasia. </li></ol><img alt="Figura 1" src="/files/ftp_upload/53045/53045fig1highres.jpg" width="700" /> Figura 1. Schema di processo globale. Vedere il testo per i dettagli. <a href="https://www.jove.com/files/ftp_upload/53045/53045fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a>

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The authors have nothing to disclose.

Nel nostro attuale protocollo, TiO 2 film è stato formato da RF-magnetron sputtering. Favoriamo questo metodo di deposito in quanto ci permette di preparare in modo riproducibile un fotocatalitico TiO 2 film con una rugosità sub-nm. Anche se i processi di deposizione sputtering sono familiari a scienziati dei materiali e ingegneri elettronici, potrebbe non essere abbastanza accessibile per i biologi. In tal caso, TiO 2 film di spin-rivestite sarebbe una scelta alternativa 23.</sup...

Processi litografia di semiconduttori e suoi derivati ​​- come fotolitografia 1,2, litografia a fascio elettronico 3-6, 7-10 e stampare microcontact - sono diventati uno strumento stabilito in biologia cellulare crescere cellule viventi in una posizione e geometria definita. Il metodo patterning si basa sull&#39;utilizzo di substrati microfabbricati, costituito da micro-isola di cellule permissive rivestimento in uno sfondo non permissive. Tale substrato serve come modello per modello cellule. Queste tecnologie ci hanno fornito i nuovi metodi per progettare cellule e la loro funzione a livello singolo e multi-cellulari, per estrarre le proprietà intrinseche delle cellule, e per aumentare la produttività di base cellulare di screening farmaco 11. Il grado di libertà in patterning cellula sarebbe aumentare notevolmente se la geometria motivo template potrebbe essere modificato in situ, cioè, mentre le cellule sono coltivate sulle surface. I metodi convenzionali per la formazione di pattern non possono essere applicati direttamente qui, dato che elaborano campioni in atmosfera o in vuoto. Sono stati proposti quindi varie nuove tecniche di modificazione superficiale, che si basano, per esempio, su composti fotoreattivi 12,13 o ablazione laser 5,14, solo per citarne alcuni. I metodi proposti sono stati ben recensito da Robertus et al. 15, e più recentemente da Choi et al. 16 e 17 Nakanishi. Qui, in questo articolo, si descrive un nuovo protocollo di in-situ modifica della superficie, che si avvale di decomposizione fotocatalitica di molecole organiche su un biossido di titanio (TiO2) Superficie 18,19. In questo metodo, un film TiO 2 è inserito tra il substrato di vetro e la pellicola organica che interfaccia le cellule, e la pellicola organica viene decomposta in situ irradiando localmente ultravioletta (UV)luce per una regione di interesse (λ &lt;388 nm). Abbiamo dimostrato che il nuovo protocollo può essere utilizzato per creare microdisegni di proteine ​​della matrice extracellulare e le cellule viventi sia ex situ e in situ. TiO 2 è biocompatibile, chimicamente stabile, e otticamente trasparente, cui caratteristiche rende adatto ad introdurre in esperimenti di coltura cellulare. Questo protocollo fornisce una scienza dei materiali a base alternativa per modificare ponteggi coltura cellulare in ambiente coltura cellulare.

<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

Contaminanti organici adsorbiti sulla superficie del biossido di titanio (TiO 2) possono essere decomposti da fotocatalisi sotto ultravioletta (UV). Qui si descrive l&#39;impiego di TiO 2 fotocatalisi alterare localmente affinità cellule della superficie del substrato un nuovo protocollo. Per questo esperimento, un sottile TiO 2 film era sputtering rivestite su un vetrino di vetro, e la superficie TiO 2 è stato successivamente modificato con un monostrato silano organico derivato da octadecyltrichlorosilane (OTS), che inibisce l&#39;adesione cellulare. Il campione è stato immerso in un mezzo di coltura cellulare, e concentrato luce UV è stata irradiata ad una regione ottagonale. Quando un neuronali PC12 linea cellulare sono state piastrate sul campione, cellule rispettati solo sulla zona UV-irradiati. Mostriamo inoltre che tale modificazione superficiale può essere eseguita anche in situ, cioè, anche quando le cellule crescono sul substrato. Modifica corretta della superficie necessaria una matrice extracellulare protein collagene di essere presente nel mezzo al momento della irradiazione UV. La tecnica qui presentata può potenzialmente essere impiegato in tipi di cellule patterning più per la costruzione di sistemi di co-coltura o di manipolare arbitrariamente cellule in coltura.

Figura 2A mostra un&#39;immagine in sezione microscopia elettronica a scansione (SEM) della TiO 2 film di sputtering depositato. Dall&#39;osservazione, spessore del film è stata stimata essere di circa 150 nm. Notevole ecco la planarità del depositato TiO 2 film. Ulteriori analisi mediante microscopia a forza atomica (AFM) ha rivelato che la radice quadrata media (RMS) rugosità della superficie era di 0,2 nm (Figura 2B). Quando la su...

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.

Photopatterning proteine ​​e cellule in acquosa ambiente utilizzando TiO<sub&gt; 2</sub&gt; Fotocatalisi

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 Views.  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.

Fabbricazione di elettrochimica-DNA biosensori per il rilevamento Reagentless di acidi nucleici, proteine ​​e piccole molecole

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. ...

Ricerca

Bioingegneria

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.

Visualizzazione proteine ​​e complessi macromolecolari di Negative EM Stain: dalla preparazione alla griglia Image Acquisition

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

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.

Un Toolkit per permettere la conversione di idrocarburi in ambienti acquosi

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.

Time-lapse imaging di fluorescenza della crescita Root Arabidopsis con manipolazione accelerato dell&#39;ambiente root usando il 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, ...

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.

Cella Co-cultura Patterning Uso acquose due sistemi monofase

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.

Trapianto di progenitori miogenici pluripotenti derivati da cellule staminali indotte simili a mesoangioblasti nei modelli di topo di rigenerazione muscolare

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.

Luminescence Resonance Energy Transfer allo Studio conformazionale Cambiamenti nella membrana proteine ​​espresse in cellule di mammifero

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.

Acquose goccioline utilizzate come microreattori enzimatica e loro attuazione elettromagnetica

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.

Un metodo versatile di patterning proteine ​​e cellule

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

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group&#8217;s method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

Here, we present a protocol to produce water soluble recombinant spider silk protein solutions and the material forms that can be formed from those solutions.

Formazione del materiale di sete di ragno ricombinante attraverso la Solvazione acquosa utilizzando calore e pressione

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order ...

Photopatterning proteine ​​e cellule in acquosa ambiente utilizzando TiO

Photopatterning proteine e cellule in acquosa ambiente utilizzando TiO