Agradecemos a los Dres. John Hancock, Ray Grill, Andrew Morris y Olga Chumakova por su apoyo. El FlexStation 3 sistema es parte del Fondo de imágenes Cytodynamic UTHSC. También queremos agradecer a los Dres. Ardem Patapoutian y Michael Bandell del Instituto de Investigación Scripps y del Instituto de Genómica de la Fundación de Investigación Novartis (GNF) para proporcionar la línea celular HEK293-hTRPA1 estable. La investigación fue apoyada en parte por subvenciones del NIH (GM081658 a MXZ) y Centro Médico de Texas Centro de Enfermedades Digestivas (a HH) y la Misión de conexión / TIRR Fundación (a HH).

1. Preparaciones de células en placas de 96 pozos Para las células HEK293 transitoria trasfected con cDNA TRPA1 humanos <ol><li> HEK293 células se cultivan para uno o dos días en medio esencial mínimo de Dulbecco (DMEM) que contiene 4,5 mg / ml de glucosa (Thermo Scientific, SH3002201), suplementado con 10% inactivado por calor suero fetal bovino (Invitrogen, 16000-044), 50 unidades / ml de penicilina y 50 mg / ml de estreptomicina (Invitrogen, 15140-163). En el día de la transfección, la densidad celular debe llegar a un 70-90%. </li><li> Escudo placas de 96 pozos con la pipeta 50 l poli-L-ornitina (Sigma P3655, 20 mg / ml en agua destilada estéril) a cada pocillo e incubar a 37 ° C durante al menos 15 min. Enjuague bien cada vez con fosfato de Dulbecco solución salina tamponada (DPBS) sin Mg 2 + y Ca 2 + (Thermo Scientific, SH3002803). La placa se puede dejar en la campana de cultivo celular por unas horas. </li><li> Por cada pocillo de una placa de 96 pozos, se diluye en un tubo Eppendorf de 1,5 ml 0,2 mg cDNA en 25 l OPTI-MEM (Invitrogen, 31985-070). Ampliar proporcionalmente en función del número de pozos que se transfectadas por el mismo cDNA. También diluir en un aparte de 1,5 ml tubo Eppendorf 0,4 l Lipofectamine 2000 (Invitrogen, 11668-019) en 25 l OPTI-MEM para cada pozo que se transfectadas. Esta escala de acuerdo a la cantidad total de pozos que se transfectadas. </li><li> Agitar brevemente el 2000 cDNA diluido y Lipofectamine y dejar que ellos se sientan en la temperatura ambiente durante unos 5 minutos. </li><li> La transferencia de un volumen igual de la Lipofectamine diluida 2000 al tubo que contiene el cDNA diluido vórtice, y deja reposar la mezcla a temperatura ambiente durante horas 15 minutos a 2. Durante este tiempo, la transferencia del cDNA / Lipofectamine 2000 mezcla a 49 l / pocillo de la placa recubierta con poli-L-ornitina. </li><li> Trypsinize las células HEK293 (Paso 1.1), cuenta la densidad de células utilizando un hemocitómetro o un contador de células automatizado Condesa (Invitrogen, C10227), y la transferencia de una cantidad deseada de células (generalmente alrededor de ~ 125,000-150,000 células / pocillo) a una estéril de 15 ml cónico fondo del tubo de centrífuga. Centrifugar a 1000 rpm x 5 min. Resuspender las células en ~ 1,250,00-1,500,000 células / ml en el medio de cultivo DMEM, suplementado con inactivado por calor, 10% de suero bovino fetal, pero sin antibióticos. </li><li> Dispensar 98 l de suspensión celular a cada pocillo que contiene el cDNA-Lipofectamine 2000 utilizando una pipeta de mezcla multicanal o el dispensador de microplacas microrrelleno (Bio-Tek). Deje que la placa de sentarse en el capó, por lo menos 30 minutos antes de colocarlo a la humidificado incubadora de cultivo celular. Se incuba a 37 ° C CO, 5% 2 de 20 a 44 hrs. </li></ol> Para HEK293 células que expresan establemente TRPA1 <ol start="8"><li> Siga el paso 1.2 para cubrir placas de 96 pozos. </li><li> La línea inducible HEK293-hTRPA1 celular fue generosamente proporcionado por el Dr. A. Patapoutian (The Scripps Research Institute) y se mantiene en el mismo medio que el de tipo salvaje células HEK293 (Paso 1.1), pero suplementado con 5 mg / ml blasticidin (Invitrogen, A1113902) y 50 ug / ml de higromicina B (Invitrogen, 10687010). Cuando la densidad celular alcanza alrededor del 90%, trypsinize células, cuenta la densidad de células y centrifugar la cantidad deseada de las células a 1000 rpm x 5 min. Resuspender las células a una densidad de ~ 1.000.000 de células / ml en el mismo medio de cultivo complementado con doxiciclina (Fisher, BP26535, 0,25 g / ml). </li><li> Prescindir de la suspensión celular a poli-L-ornitina placas recubiertas de 100 l / pocillo utilizando una pipeta de canales múltiples o la microrrelleno (Bio-Tek). Deje que la placa de sentarse en el capó, por lo menos 30 minutos antes de colocarlo a la humidificado incubadora de cultivo celular. Se incuba a 37 ° C, 5% de CO2 durante 16 a 24 horas. </li></ol> 2. Soluciones <ol><li> Tampón de Hank solución de sal (HBSS) se utiliza como la solución extracelular. Que contiene (en 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 glucosa, 10 HEPES, con pH ajustado a 7,4 con NaOH 1 N). </li><li> Fluo-4 tampón de ensayo contiene 2 mM probenecid en HBSS. Hacer 250 mM de valores probenecid (Sigma, P8761) en 0,5 N NaOH. Diluir a HBSS en una proporción de 1:125. Reajustar el pH a 7.4 con 1 N HCl. </li></ol> 3. Célula de carga con Fluo-4 AM <ol><li> Disolver 1 mg Fluo-4 AM (Invitrogen, F14202) en DMSO anhidro 912 l para hacer una solución madre de 1 mM. El Fluo-4 AM solución de reserva se pueden almacenar a -20 ° C durante al menos un mes. </li><li> Se mezclan 10 l 1 mM Fluo-4 AM con 10 l Pluronic F-127 (Invitrogen, P3000MP, el 20% (w / v) en DMSO) y luego transferir todo el contenido a 5 ml de tampón de ensayo de Fluo-4 (Paso 2.2) complementado con la especie bovina 0,1% de albúmina sérica (Sigma, A9418). </li><li> Lavar las células cultivadas en placas de 96 pozos (Paso 1.7 o 1.10) con HBSS a 80 l / y 2 veces con un lavador de microplacas (como ELx405TM de Bio-Tek). </li><li> Agregue el Fluo-4 AM carga solución (Paso 3.2) a 50 l / pocillo utilizando una pipeta de canales múltiples o la microrrelleno (Bio-Tek) y se incuba la placa a 37 ° C durante 1 hora. </li><li> Lavar las células 3 veces con el tampón de ensayo Fluo-4 (Paso 2.2.) Usando el lavador de microplacas ELx405TM. Después del último lavado, deje 80 l del tampón de Fluo-4 del ensayo en cada pocillo. </li></ol> 4. Preparación de la placa de compuesto <ol><li> Durante el período de 1 hora de carga celular, preparar una serie de diluciones de los agonistas de 3x (o antagonistas) de 3 veces de la concentración final deseada en el buffer de Fluo-4 ensayo. </li><li> Pipeta de 250 a 300 l de las soluciones de compuesto diluido en un plato compuesto de 96 pozos. Haga arreglos para que las diluciones seriadas de un compuesto y los correspondientes controles positivos y negativos en la misma columna de la placa compuesto siempre que sea posible, porque FlexStation 3 dice una sola columna a la vez. </li></ol> 5. Placa de la lectura en FlexStation 3 <ol><li> Encienda el sistema (FlexStation 3, la computadora y el software SoftMax Pro 5.2) </li><li> Guardar el experimento con un nuevo nombre de archivo. En el set-up, elegir el modo de FLEX y escriba los siguientes valores: </li></ol> Protocolo n º 1, el efecto agonista <img src="/files/ftp_upload/3149/3149table1.jpg" alt="Tabla 1" /> Protocolo n º 2, el efecto antagonista <img src="/files/ftp_upload/3149/3149table2.jpg" alt="Tabla 2" /><ol start="3"><li> Coloque una caja nueva punta, compuesto de placa y la placa de la lectura de las bandejas adecuadas FlexStation 3 y haga clic en botón &quot;Leer&quot;. El FlexStation 3 leerá una columna a la vez y añadir compuestos en los puntos de tiempo preprogramado sin interrumpir la lectura. Se tarda unos 3 minutos (5 minutos para el Protocolo n º 2) para terminar de leer una columna y la máquina continuará a leer la siguiente columna con adiciones compuesto cabo según lo programado por el usuario en el paso 5.2 hasta que todas las columnas se leen. </li></ol> 6. Análisis de los datos <ol><li> Los datos experimentales pueden ser procesados ​​directamente a través de la SoftMax Pro 5.2 software o copiar y pegar en cualquier programa de hoja de cálculo, como Microsoft Excel. Concentración de la curva de respuesta de un agonista del ácido flufenámico TRPA1 (FFA) se construye con los siguientes mínimos cuadrados rutina de instalación. <img src="/files/ftp_upload/3149/3149eq1.jpg" alt="La ecuación 1" /> , Donde Y es la respuesta observada, Y max es la respuesta máxima normalizada, C es la concentración del fármaco correspondiente, la CE 50 es la concentración que provocó una respuesta mitad de la máxima, y H n es el coeficiente de Hill aparente. La concentración de inhibición de la curva de un compuesto antagonista de los supuestos TRPA1 A se obtiene mediante el uso de una concentración fija de FFA (100 M), y progresivamente el aumento de la concentración del compuesto A. IC 50 valor del compuesto A se calcula por mínimos cuadrados ajuste a las siguientes ecuación. <img src="/files/ftp_upload/3149/3149eq2.jpg" alt="La ecuación 2" /> , Donde Y es la respuesta observada y Y max se normaliza la respuesta de pico, [Ant] es el correspondiente compuesto una concentración, el IC50 es la concentración de antagonista que produce la mitad de la máxima inhibición de la FFA, potenciales evocados, y n H es la colina de aparente coeficiente. </li></ol> 7. Los resultados representativos: TRPA1 es un Ca 2 +-permeable canal catiónico, la activación de lo que lleva a la entrada de calcio que puede ser detectada por el Ca 2 + sensibles al medio de contraste, Fluo-4 (1-2). Una línea celular HEK293-hTRPA1 estable se utilizó para estudiar la relación concentración-respuesta de TRPA1 agonistas y antagonistas. Las células fueron cargadas con Fluo-4 AM, ubicada en 80 l del tampón de ensayo, y leer con FlexStation 3. Después de grabar la fluorescencia basal durante 30 s, 40 l de una dilución en serie de los agonistas de TRPA1, FFA, cada uno a 3 veces de la concentración final deseada, se añadieron a las células a través de una pipeta de varios canales incluye como parte del módulo de fluidos de FlexStation 3. Los cambios de fluorescencia fueron controlados por un adicional de 180 s. Los cambios relativos Fluo-4 de fluorescencia para cada pozo se expresaron como Df = (F - F 0), donde 0 F y F son los valores de fluorescencia en el momento de interés y el comienzo de la lectura, respectivamente. Rastros representante de los cursos a tiempo a los cambios de fluorescencia en respuesta a una serie de concentraciones de ácidos grasos libres se muestra en la figura. 1. Nota para los últimos cuatro concentraciones de ácidos grasos libres, aunque los niveles de respuesta máxima son similares, la cinética de activación es claramente más rápido en el más alto que en las concentraciones más bajas de ácidos grasos libres. Por lo tanto, para distinguir estas diferencias, la concentración - relación dosis-respuesta fue construido por la medición de la tasa inicial (pendiente) del incremento de fluorescencia tras la adición de diferentes concentraciones de ácidos grasos libres (Fig. 2). El efecto máximo medioconcentración ive (CE 50) de la FFA se determinó que 55,4 M (n = 3). Para probar la supuesta TRPA1 antagonista, el compuesto A, se realizó un experimento similar utilizando el protocolo n º 2 (Paso 2). En este caso, una serie de diluciones del antagonista se añadieron a los 30 s (40 l de 3x deseada concentración final) y los ácidos grasos libres se añadió más tarde 180 s (60 l menos 300 M para una concentración final de 100 mM). Compuesto A dependiente de la dosis reduce la respuesta de las células de TRPA1 FFA con una media máxima concentración inhibitoria (IC50) de 4,3 M (n = 3) (Fig. 2). <img src="/files/ftp_upload/3149/3149fig1.jpg" alt="Figura 1"/> Figura 1. Concentración-dependiente de la activación de TRPA1 humanos por parte de la FFA. Las huellas son aumentar la FFA evocados de la fluorescencia en el tiempo (las respuestas sólo en los primeros 60 s se muestran para ilustrar mejor la rápida cinética de las respuestas evocadas por FFA). Tenga en cuenta que a mayores concentraciones de FFA evocado incremento de fluorescencia con una. Cinética mucho más rápido que en concentraciones más bajas, a pesar de que los niveles de fluorescencia máxima fueron similares <img src="/files/ftp_upload/3149/3149fig2.jpg" alt="Figura 2"/> Figura 2 Concentración -. Relaciones de respuesta de FFA el cambio inducido por fluorescencia y el compuesto A-inducida por la inhibición de la respuesta de la FFA en humanos TRPA1 expresado de forma estable en células HEK293. Δ, la concentración - relación dosis-respuesta de la FFA se determinó mediante la medición de la tasa inicial (pendiente) del incremento de fluorescencia normalizada con la adición de diferentes concentraciones de ácidos grasos libres. O, de concentración-respuesta de la inhibición de la FFA-respuesta evocada del calcio por una supuesta TRPA1 antagonista, compuesto A.

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Intracelular de Ca 2 + juega un papel importante en muchas condiciones fisiológicas y patológicas, como la liberación de neurotransmisores (3), potencial de acción cardíaco (4), la contracción de las células musculares (5), la transducción de señal de celular (6-7), y la muerte celular excitotóxica ( 8). La medición de Ca 2 + intracelular niveles de ayuda a dilucidar los cambios funcionales de Ca 2 +-permeables los canales en la membrana plasmática o organelos intracelulares ...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> FlexStation 3 </td><td> Molecular Devices </td><td> FLEX3 </td><td></td></tr><tr><td> 96-Bueno, Consejos FlexStation Pipet </td><td> Molecular Devices </td><td> 9000-0912 </td><td></td></tr><tr><td> Placas de 96 pozos </td><td> Greiner Bio-One </td><td> 655090 </td><td></td></tr><tr><td> 96-Bueno, claro placas de fondo plano </td><td> Costar </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-4 AM </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> Albúmina de suero 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

El FlexStation Molecular Devices &quot;3 es una mesa de trabajo multi-modo de lector de microplacas capaz de medir la fluorescencia automatizado de múltiples placas. Es ideal para medianas y de alto rendimiento de las pantallas en el ámbito académico. Tiene un módulo de transferencia de fluidos integrado equipado con una pipeta de canales múltiples y la máquina lee una columna a la hora de controlar los cambios de fluorescencia de una variedad de reactivos fluorescentes. Por ejemplo, FlexStation 3 se ha utilizado para estudiar la función de Ca 2 +-permeables los canales de iones y proteínas G-receptores acoplados mediante la medición de los cambios intracelulares de Ca 2 + libre los niveles. Transitoria receptor potencial (TRP) los canales son una gran familia de canales de cationes no selectivos que juegan un papel importante en muchas funciones fisiológicas y fisiopatológicas. La mayoría de los canales TRP son el calcio permeable e inducir la entrada de calcio a la activación. En este video, se demuestra la aplicación de FlexStation 3 para estudiar el perfil farmacológico de los canales TRPA1, un sensor molecular de numerosos estímulos nocivos. HEK293 transitoria o estable expresando humanos TRPA1 canales, que se cultiva en placas de 96 pozos, se cargan con una Ca 2 + sensibles tinte fluorescente, Fluo-4, y en tiempo real los cambios de fluorescencia en estas células se midió antes y durante la aplicación de un agonista de la TRPA1 utilizando el modo de flexión de la FlexStation 3. El efecto de un antagonista de los supuestos TRPA1 también fue examinado. Los datos se transfieren desde el software Pro SoftMax para construir relaciones de concentración-respuesta de TRPA1 activadores e inhibidores.

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

Este video ofrece un protocolo detallado para estudiar el perfil farmacológico de humanos TRPA1 canales a través de FlexStation 3. El protocolo cubre los detalles de la preparación de la célula, la carga de tinte y el funcionamiento del lector de microplacas, FlexStation 3.

Basados ​​en células de calcio de ensayo para el medio a cribado de alto rendimiento de las funciones del PRT canal utilizando 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 vistas.  The University of Texas Health Science Center at Houston. Este video ofrece un protocolo detallado para estudiar el perfil farmacológico de humanos TRPA1 canales a través de FlexStation 3. El protocolo cubre los detalles de la preparación de la célula, la carga de tinte y el funcionamiento del lector de microplacas, 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 de polímeros de alto rendimiento para el descubrimiento de Biomateriales

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

Investigación

Bioingeniería

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) para la selección de alto rendimiento antifúngico

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.

Proteínas de alto rendimiento del generador de expresiones utilizando una plataforma de microfluidos

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.

Generación de Scalable, metálico de alta Aspect Ratio nanocompuestos en una Biológica Medio Líquido

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 protocolo para la de alto rendimiento automatizado tomografía crioelectrónica

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.

De alto rendimiento de identificación de bacterias polímeros repelentes para dispositivos médicos

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 para la terapia regenerativa inyectable y drogas de alto rendimiento

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.

Optimización de procesos mediante microbiorreactores automatizados de alto rendimiento en el cultivo de células de ovario de hámster chino

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.

Identificación de alto rendimiento de la resistencia a Pseudomonas syringae pv. Tomate en tomate usando el ensayo de inundación de plántulas

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.

Generación de condiciones ambientales dinámicas utilizando un dispositivo microfluídico de alto rendimiento

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

Basados ​​en células de calcio de ensayo para el medio a cribado de alto rendimiento de las funciones del PRT canal utilizando FlexStation 3

Basados en células de calcio de ensayo para el medio a cribado de alto rendimiento de las funciones del PRT canal utilizando FlexStation 3