Ringraziamo Drs. John Hancock, Ray Grill, Andrew Morris e Olga Chumakova per il loro supporto. Il sistema a 3 FlexStation fa parte dello strumento UTHSC Imaging Cytodynamic. Ringraziamo anche Drs. Ardem Patapoutian e Michael Bandell dalla Scripps Research Institute e del Genomics Institute della Novartis Research Foundation (GNF) per fornire la linea cellulare HEK293-hTRPA1 stabile. La ricerca è finanziata in parte dalle concessioni dal NIH (GM081658 a MXZ) e Texas Medical Center Digestive Diseases Center (a SS) e Mission Connect / TIRR Foundation (a SS).

1. Preparazioni di cellule in piastre da 96 pozzetti Per le cellule HEK293 transitoriamente trasfected con TRPA1 umano cDNA <ol><li> HEK293 cellule sono coltivate per uno o due giorni in terreno minimo essenziale Dulbecco (DMEM) contenente 4,5 mg / ml di glucosio (Thermo Scientific, SH3002201), integrato con il 10% di siero siero fetale bovino (Invitrogen, 16000-044), 50 unità / ml di penicillina e 50 mg / ml di streptomicina (Invitrogen, 15140-163). Il giorno della transfezione, la densità cellulare dovrebbe raggiungere il 70-90%. </li><li> Cappotto piastre a 96 pozzetti pipettando 50 microlitri poli-L-ornitina (Sigma P3655, 20 mg / ml in acqua distillata sterile) in ciascun pozzetto e incubare a 37 ° C per almeno 15 min. Lavare ogni pozzetto una volta con fosfato Dulbecco Buffered Saline (DPBS) senza Mg 2 + e Ca 2 + (Thermo Scientific, SH3002803). La piastra può essere lasciato nel cofano coltura cellulare per qualche ora. </li><li> Per ogni pozzetto di una piastra da 96 pozzetti, diluire in 1,5 ml di tubo Eppendorf 0,2 mcg cDNA in 25 microlitri OPTI-MEM (Invitrogen, 31985-070). Scalare fino proporzionalmente in base al numero di pozzi da transfettate con il cDNA stesso. Anche diluire in un apposito 1,5 ml di tubo Eppendorf 0,4 microlitri Lipofectamine 2000 (Invitrogen, 11668-019) in 25 microlitri OPTI-MEM per ogni bene a essere transfettate. Questa scala fino in base al numero totale di pozzi per essere transfettate. </li><li> Brevemente vortice del 2000 cDNA diluito e Lipofectamine e lasciarli stare alla temperatura ambiente per circa 5 min. </li><li> Trasferire un volume uguale della Lipofectamine diluito 2000 per il tubo che contiene il cDNA diluito, vortex e lasciare che la miscela a temperatura ambiente per 15 minuti a 2 ore. Durante questo tempo, trasferire il cDNA / Lipofectamine 2000 miscela al 49 microlitri / bene al poli-L-ornitina placca rivestita. </li><li> Trypsinize le cellule HEK293 (Punto 1.1), conta la densità delle cellule utilizzando un emocitometro o contatore di cellule contessa automatizzati (Invitrogen, C10227), e trasferire una quantità desiderata di cellule (generalmente circa ~ 125,000-150,000 cellule / pozzetto) a 15 ml sterile conica con fondo provetta da centrifuga. Centrifugare a 1000 RPM x per 5 min. Risospendere le cellule a ~ 1,250,00-1,500,000 cellule / ml nel terreno di coltura DMEM, supplementato con 10% di siero siero fetale bovino, ma senza antibiotici. </li><li> Dispensare 98 microlitri di sospensione cellulare in ogni pozzetto che contiene il cDNA-Lipofectamine 2000 utilizzando una miscela pipetter multicanale o il Dispenser Microplate Microfill (Bio-Tek). Lasciate che la piastra di sedere nel cofano per almeno 30 minuti prima di metterlo alla cultura incubatore umidificato cellulare. Incubare a 37 ° C, 5% di CO 2 per 20-44 ore. </li></ol> Per le cellule HEK293 che esprimono stabilmente TRPA1 <ol start="8"><li> Seguire i passi da 1,2 a cappotto piastre a 96 pozzetti. </li><li> La linea inducibile HEK293-hTRPA1 cella era generosamente fornito dal Dr. A. Patapoutian (The Scripps Research Institute) e mantenuto nel mezzo come il wild-type cellule HEK293 (Punto 1.1) ma integrata con 5 mcg / ml blasticidin (Invitrogen, A1113902) e 50 mg / ml Hygromycin B (Invitrogen, 10687010). Quando densità cellulare raggiunge circa il 90%, trypsinize cellule, conta densità cellulare, e centrifugare la quantità desiderata di cellule a 1000 x RPM per 5 min. Risospendere le cellule ad una densità di circa 1.000.000 cellule / ml nel terreno di coltura stesso integrato con doxiciclina (Fisher, BP26535, 0,25 mg / ml). </li><li> Dispensare la sospensione cellulare rivestito di lastre di poli-L-ornitina a 100 l / pozzetto utilizzando una pipetter multicanale o il Microfill (Bio-Tek). Lasciate che la piastra di sedere nel cofano per almeno 30 minuti prima di metterlo alla cultura incubatore umidificato cellulare. Incubare a 37 ° C, 5% CO 2 per 16 - 24 ore. </li></ol> 2. Soluzioni <ol><li> Soluzione salina tamponata di Hank (HBSS) è usato come soluzione extracellulare. Esso contiene (in mm): 137 NaCl, 5.4 KCl, 0,25 Na 2 HPO 4, 0,44 KH 2 PO 4, 1,3 CaCl 2, 1,0 MgSO 4, 1,0 MgCl 2, 10 di glucosio, 10 HEPES, con pH regolato a 7,4 con NaOH 1N). </li><li> Fluo-4 tampone contiene 2 mM probenecid in HBSS. Fai 250 mm magazzino probenecid (Sigma, P8761) in 0,5 N NaOH. Diluire a HBSS con un rapporto di 1:125. Regolare il pH a 7,4 con 1 N HCl. </li></ol> 3. Celle di carico con Fluo-04:00 <ol><li> Sciogliere 1 mg Fluo-4 AM (Invitrogen, F14202) nel 912 microlitri di DMSO anidro per ottenere una soluzione stock di 1 mM. Il Fluo-4:00 soluzione madre può essere conservata a -20 ° C per almeno un mese. </li><li> Mix 10 microlitri 1 mM-Fluo 04:00 con 10 microlitri Pluronic F-127 (Invitrogen, P3000MP, 20% (w / v) in DMSO) e poi trasferire l&#39;intero contenuto di 5 ml Fluo-4 tampone (Step 2.2) integrato con 0,1% di albumina sierica bovina (Sigma, A9418). </li><li> Lavare le cellule coltivate in piastre a 96 pozzetti (passo 1,7 o 1,10) con HBSS a 80 microlitri / ben 2 volte con una rondella di micropiastre (come ELx405TM da Bio-Tek). </li><li> Aggiungere il Fluo-4 AM carico soluzione (Fase 3.2) a 50 microlitri / pozzetto utilizzando una pipetter multicanale o il Microfill (Bio-Tek) e incubare la piastra a 37 ° C per 1 ora. </li><li> Lavare le cellule 3 volte con il Fluo-4 tampone (Punto 2.2.) Utilizzando la rondella micropiastre ELx405TM. Dopo l&#39;ultimo lavaggio, lasciare 80 ml di buffer di Fluo-4 test in ciascun pozzetto. </li></ol> 4. Preparazione del piatto composto <ol><li> Durante il periodo 1 cella di carico ora, preparare una serie di diluizioni 3x di agonisti (o antagonisti) di 3x delle concentrazioni finale desiderato nel buffer di Fluo-4 test. </li><li> Pipetta 250-300 ml di soluzioni composto diluito ad una piastra composto da 96 pozzetti. Provvede alla traduzione della diluizioni seriali di un composto e dei controlli positivo e negativo corrispondente nella stessa colonna del piatto composto ogni volta che è possibile perché FlexStation 3 recita una colonna alla volta. </li></ol> 5. Di lettura targhe in FlexStation 3 <ol><li> Accendere il sistema (FlexStation 3, computer e il SoftMax Pro 5.2 del software) </li><li> Salva l&#39;esperimento con un nuovo nome. Al set-up, scegliere la modalità FLEX e immettere i seguenti valori: </li></ol> Protocollo n. 1, effetto agonista <img src="/files/ftp_upload/3149/3149table1.jpg" alt="Tabella 1" /> Protocollo n. 2, effetto antagonista <img src="/files/ftp_upload/3149/3149table2.jpg" alt="Tabella 2" /><ol start="3"><li> Inserire una nuova casella punta, piatto composto e la piastra di lettura per i vassoi appropriato in FlexStation 3 e fare clic sul pulsante &quot;leggere&quot;. Il FlexStation 3 sarà letta una colonna alla volta e aggiungere composti nei punti di tempo programmato senza interrompere la lettura. Ci vogliono circa 3 minuti (5 min per il protocollo # 2) per finire di leggere una colonna e la macchina continua a leggere la colonna successiva, con aggiunte composto eseguito come programmato dall&#39;utente al passo 5,2 fino a quando tutte le colonne vengono letti. </li></ol> 6. Analisi dei dati <ol><li> I dati sperimentali possono essere trattati direttamente con il SoftMax Pro 5.2 del software o copiato e incollato in qualsiasi programma di foglio di calcolo, come Microsoft Excel. Curva concentrazione-risposta di un acido TRPA1 agonista flufenamic (FFA), è costruito utilizzando i seguenti minimi quadrati routine di montaggio. <img src="/files/ftp_upload/3149/3149eq1.jpg" alt="Equazione 1" /> , Dove Y è la risposta osservata, Y max è la risposta normalizzata massima, C è la concentrazione del farmaco corrispondente, CE 50 è la concentrazione che evoca una mezza massima risposta, e n H è il coefficiente di Hill apparente. Concentrazione di inibizione curva per un presunto TRPA1 composto antagonista A è ottenuta utilizzando una concentrazione fissa di FFA (100 micron), e progressivamente aumentando la concentrazione di A. composto IC 50 Valore del composto A si calcola minimi quadrati al seguente equazione. <img src="/files/ftp_upload/3149/3149eq2.jpg" alt="Equazione 2" /> , Dove Y è la risposta osservata e Y max è normalizzata risposta di picco, [Ant] è il composto corrispondente A di concentramento, IC 50 è la concentrazione di antagonista che produce la metà-massimale inibizione della FFA-risposta evocata e n H è la collina apparente coefficiente. </li></ol> 7. Rappresentante dei risultati: TRPA1 è un +-permeabile canale catione Ca 2, l&#39;attivazione dei quali porta a afflusso di calcio che può essere rilevato dal Ca 2 +-sensitive colorante, Fluo-4 (1-2). Una linea cellulare HEK293-hTRPA1 stabile è stato utilizzato per studiare la concentrazione-risposta di TRPA1 agonisti e antagonisti. Le cellule sono state caricate con Fluo-4 AM, posto in 80 microlitri della tampone, e leggere con FlexStation 3. Dopo aver registrato la fluorescenza di base per 30 s, 40 ml di una diluizione di serie del agonista TRPA1, FFA, ciascuno a 3 volte la concentrazione finale desiderata, sono stati aggiunti alle cellule attraverso un multi-canale pipetter incluso come parte del modulo fluidica di FlexStation 3. I cambiamenti fluorescenza sono stati monitorati per ulteriori 180 s. Le variazioni relative Fluo-4 fluorescenza per ciascun pozzetto sono stati espressi come ΔF = (F - F 0), dove 0 F e F sono i valori di fluorescenza, al momento di interesse e l&#39;inizio della lettura, rispettivamente. Tracce rappresentante dei corsi tempo ai cambiamenti fluorescenza in risposta ad una serie di concentrazioni di FFA sono mostrati in fig. 1. Nota per le ultime quattro concentrazioni di FFA, anche se i livelli di risposta massima sono simili, la cinetica di attivazione è chiaramente più veloce al superiore alle concentrazioni inferiori FFA. Pertanto, per distinguere queste differenze, la concentrazione - relazione dose-risposta è stato costruito misurando i tassi iniziali (pendenza) di aumento di fluorescenza dopo l&#39;aggiunta di varie concentrazioni di FFA (Fig. 2). L&#39;effetto mezzo massimoconcentrazione ive (CE 50) di FFA è stato determinato in 55,4 mM (n = 3). Per testare la putativo TRPA1 antagonista, composto A, abbiamo effettuato un esperimento simile con protocollo n. 2 (punto 2). In questo caso, una serie di diluizioni del antagonista sono stati aggiunti a 30 s (40 ml di 3x concentrazione finale desiderata) e il FFA è stato aggiunto dopo 180 s (60 microlitri a 300 mM per una concentrazione finale di 100 mM). Composto A dose-dipendente ridotto la risposta delle cellule TRPA1 di FFA con una mezza massima concentrazione inibente (IC 50) di 4,3 mM (n = 3) (Fig. 2). <img src="/files/ftp_upload/3149/3149fig1.jpg" alt="Figura 1"/> Figura 1. Concentrazione-dipendente l&#39;attivazione di TRPA1 umani da parte di FFA. Le tracce sono aumentare FFA-evocato di fluorescenza nel tempo (risposta solo nei primi 60 s vengono mostrati per illustrare meglio la cinetica veloce del FFA-evocate risposte). Si informa che alle alte concentrazioni di FFA evocato aumento di fluorescenza con una cinetica molto più veloce rispetto a concentrazioni inferiori, anche se i livelli di picco di fluorescenza erano simili. <img src="/files/ftp_upload/3149/3149fig2.jpg" alt="Figura 2"/> Figura 2 Concentrazione -. Relazioni risposta per FFA cambiamento indotto fluorescenza e composto A indotta inibizione della risposta FFA umani TRPA1 stabilmente espressi in cellule HEK293. Δ, la concentrazione - relazione dose-risposta di FFA è stata determinata misurando il tasso iniziale (pendenza) di aumento di fluorescenza normalizzata dopo l&#39;aggiunta di varie concentrazioni di FFA. O, concentrazione-risposta di inibizione della FFA-evocata risposta di calcio da un presunto TRPA1 antagonista, composto A.

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Intracellulare di Ca 2 + gioca un ruolo importante in molte condizioni fisiologiche e patologiche, quali rilascio dei neurotrasmettitori (3), potenziale d&#39;azione cardiaco (4), la contrazione delle cellule muscolari (5), trasduzione del segnale cellulare (6-7), e la morte cellulare eccitotossico ( 8). Misura di Ca 2 + intracellulare livelli aiuta a chiarire cambiamenti funzionali di Ca 2 +-permeabile canali nella membrana plasmatica o organelli intracellulari e il contributo di ...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> FlexStation 3 </td><td> Molecular Devices </td><td> FLEX3 </td><td></td></tr><tr><td> 96-Beh, FlexStation Pipettare Consigli </td><td> Molecular Devices </td><td> 9000-0912 </td><td></td></tr><tr><td> Piastre a 96 pozzetti </td><td> Greiner Bio-One </td><td> 655090 </td><td></td></tr><tr><td> 96-ben chiaro piastre a fondo piatto </td><td> Co-star </td><td> 3595 </td><td></td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 12430-104 </td><td></td></tr><tr><td> Opti-MEM </td><td> Invitrogen </td><td> 31985-070 </td><td></td></tr><tr><td> Lipofectamine 2000 </td><td> Invitrogen </td><td> 11668-019 </td><td></td></tr><tr><td> Fluo, 04:00 </td><td> Invitrogen </td><td> F14202 </td><td></td></tr><tr><td> PluronicF-127 </td><td> Invitrogen </td><td> P3000MP </td><td></td></tr><tr><td> Poli-L-ornitina </td><td> Sigma </td><td> P36551 </td><td></td></tr><tr><td> probenecid </td><td> Sigma </td><td> P8761 </td><td></td></tr><tr><td> Albumina di siero bovino </td><td> Sigma </td><td> A9418 </td><td></td></tr></tbody></table>

cell-based calcium assay for medium to high throughput screening of trp channel functions using flexstation 3

FlexStation il Molecular Devices &#39;3 è un banco di lavoro multi-mode capaci di lettore per micropiastre automatico di misurazione della fluorescenza multi-pozzetti. E &#39;ideale per medie e high-throughput schermi in ambienti accademici. Ha un modulo integrato di trasferimento del liquido dotato di un multi-canale pipetter e la macchina legge una colonna alla volta per monitorare le variazioni di fluorescenza di una varietà di reagenti fluorescenti. Per esempio, FlexStation 3 è stato usato per studiare la funzione di Ca 2 +-permeabile canali ionici e G-proteine ​​recettori accoppiati misurando le variazioni di libero intracellulare di Ca 2 + livelli. Transitori recettore potenziale (TRP) i canali sono una grande famiglia di canali di comunicazione non selettivi che svolgono un ruolo importante in molte funzioni fisiologiche e fisiopatologiche. La maggior parte dei canali TRP sono il calcio permeabili e indurre influsso di calcio al momento dell&#39;attivazione. In questo video, dimostriamo l&#39;applicazione di FlexStation 3 a studiare il profilo farmacologico del canale TRPA1, un sensore molecolare per numerosi stimoli nocivi. Cellule HEK293 transitoriamente o stabilmente esprimere umano TRPA1 canali, coltivate in piastre a 96 pozzetti, vengono caricati con Ca 2 +-sensitive colorante fluorescente, Fluo-4, e le modifiche fluorescenza in tempo reale in queste cellule sono misurati prima e durante l&#39;applicazione di un agonista TRPA1 utilizzando la modalità FLEX del FlexStation 3. L&#39;effetto di un putativo TRPA1 antagonista stato anche esaminato. I dati vengono trasferiti dal software SoftMax Pro per la costruzione di concentrazione-risposta di TRPA1 attivatori e inibitori.

Watch this Scientific Journal Video about Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3 at JoVE.com

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

Questo video fornisce un protocollo dettagliato per lo studio del profilo farmacologico di umano TRPA1 canali utilizzando FlexStation 3. Il protocollo comprende dettagli di preparazione delle cellule, il caricamento colorante e il funzionamento del lettore per micropiastre, FlexStation 3.

Saggio basato su cellule di calcio per le medie e High Throughput Screening del PRT Funzioni canale utilizzando FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

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21.0K Views.  The University of Texas Health Science Center at Houston. Questo video fornisce un protocollo dettagliato per lo studio del profilo farmacologico di umano TRPA1 canali utilizzando FlexStation 3. Il protocollo comprende dettagli di preparazione delle cellule, il caricamento colorante e il funzionamento del lettore per micropiastre, FlexStation 3.

Video: Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

Microarrays polimerici per Discovery High Throughput di Biomateriali

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

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Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Candida albicans Biofilm Chip (Ca BChip) per High-throughput screening farmaco antifungino

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

High-throughput generatore di espressione delle proteine ​​Utilizzando una piattaforma microfluidica

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 &#176;C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Here we present a protocol to synthesize novel, high-aspect ratio biocomposites under biological conditions and in liquid media. The biocomposites scale from nanometers to micrometers in diameter and length, respectively. Copper nanoparticles (CNPs) and copper sulfate combined with cystine are the key components for the synthesis.

Generazione di scalabili, Metallic Tacchi-Aspect nanocompositi Rapporto in un Biologica Liquid Media

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

Usando Tomoauto: un protocollo per High-throughput automatizzato Cryo-elettrone Tomografia

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

High-throughput identificazione di batteri Polimeri repellente per dispositivi medici

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

Microtissues 3D per la terapia rigenerativa iniettabile e droga di High-throughput Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain selection and by developing bioprocess parameters. Shake flasks have been used for this purpose. They, however, lack the capability to control the process parameters such as pH and dissolved oxygen (DO). This limitation can be overcome with the help of an automated micro-bioreactor. These bioreactors mimic cultivation at a larger scale. One of the major advantages of this system is the integration of the Design of Experiment (DOE) in the software. This integration enables establishing a design where multiple process parameters can be varied simultaneously. The critical process parameters and optimum bioprocess conditions can be analyzed within the software. The focus of the work presented here is to introduce the user to the steps involved in process design in the software and incorporation of the DOE within the cultivation run.

Here, we present a detailed procedure to run a Design of Experiment in an automated micro-bioreactor followed by cell harvest and protein quantification using a Protein A column.

Ottimizzazione dei processi mediante microreattori automatizzati ad alta velocità effettiva nella coltivazione delle cellule ovaio dell'ammasso cinese

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

Identificazione ad alto contenuto di velocità effettiva della resistenza a Pseudomonas syringae pv. Pomodoro nel pomodoro utilizzando Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Generazione di condizioni ambientali dinamiche utilizzando un dispositivo microfluidico ad alta produttività

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...