Este trabajo fue financiado por el Fonds de Recherche sur la Nature et les Tecnologías de Quebec, la Fundación Canadiense para la Innovación y los Institutos Canadienses de Investigación en Salud (CIHR).

1. Microfabricación de la cabeza de la sonda (proceso que no aparecen en el video) <ol><li> Una oblea de Si-2, cuatro pulgadas de diámetro, 525 micras de espesor, con una capa de SiO2 1 m de espesor térmico es spincoated con un fotoprotector (PR) durante 45 s a 4000 rpm. </li><li> La oblea se precocidos a 110 ° C durante 50 s, y se expone a través de una máscara con todos los elementos (puertos y microcanales) durante 5 s, desarrollado y aclarado con DI. </li><li> El SiO2 descubierto es grabado en un buffer 01:07 ácido fluorhídrico (BHF), solución de ≈ 15 min (deshumectación del sustrato donde se ha grabado SiO2 indica la finalización de la etch). Un plasma de O2 o la acetona se utiliza para la ceniza o la tira del resto de PR. </li><li> Una segunda capa de PR es spin-revestido a 1500 rpm durante 45 s, dando una sobrecapa de espesor de 10 micras ≈ [31]. El patrón de SiO2 por debajo de esta capa de PR es aún visible y se utiliza para alinear la oblea con una segunda máscara con los únicos puertos. </li><li> Después de la exposición y el desarrollo de las relaciones públicas, la oblea se lava, se seca, y postbaked a 95 ° C durante 20 min. </li><li> La oblea de silicio se une a una oblea de apoyo con cera fundida blanca para proteger el plato. </li><li> Un plasma de acoplamiento inductivo (ICP) DRIE se utiliza para transferir el PR y embebidos SiO2 patrones en la topografía de obleas en un proceso de tres pasos: <ol><li> DRIE para que los puertos ≈ 500μmdeep en Si (patrón definido por el espesor PR). </li><li> Sin necesidad de descargar la oblea de la máquina de DRIE, el PR se incinera con un plasma. </li><li> La exposición SiO2 patrón actúa como máscara para un segundo proceso de grabado en seco, la creación de 50 canales m de profundidad, y la apertura del relleno y los puertos de ventilación a través de la oblea. Después de la descarga, la oblea de apoyo se separa en una corriente de agua caliente. La oblea micromecanizados se limpia con acetona, etanol y DI. </li></ol></li><li> Fichas individuales MFP son cortados en cubitos. </li><li> Un bloque de interfaz de PDMS se fabrica de fundición en un micro molde formado por dos estructurado de poli (metacrilato de metilo) (PMMA) elementos, una placa de acero pulido que forman la parte inferior, y los capilares de dos (cada una se inserta en una de las dos vías de acceso a los agujeros en la placa de acero) que sirven como marcadores de posición de los orificios de conexión de fluidos. El PDMS se cura en un horno a 60 ° C durante al menos 1 h. </li><li> El bloque de PDMS se une a una picada de chips de silicio MFP mediante la activación de las dos partes en el aire de plasma a 1 mbar durante 24 s en 230 W, y que une los dos juntos con un hecho en casa a la alineación mecánica. </li><li> El montaje se deja a los bonos en un horno de 60 º C durante un mínimo de 1 hora </li></ol> 2. Asamblea de la MFP <ol><li> Impermeable a los gases jeringas de vidrio se llenan con los reactivos adecuados utilizando jeringas y agujas de plástico para asegurarse de que no queden burbujas de aire. Por lo general, se utiliza una jeringa de 1 -10 microlitro para la inyección, y una jeringa con un volumen de 5-10 veces mayor para la aspiración. </li><li> Las jeringas están conectadas a tubos capilares con accesorios Nanotight con bajo volumen muerto. </li><li> Los capilares se llenan y se analiza las burbujas bajo el microscopio. </li><li> El chip MFP es precargadas con solución tampón para evitar la captura de burbujas cuando se conectan los vasos capilares. </li><li> Los capilares están conectados a la pieza de conexión PDMS en la cabeza de la sonda </li></ol> 3. Configuración de la impresora multifunción <ol><li> La cabeza de la sonda se sujeta en el soporte de la sonda y se monta en la estación de la sonda sobre un microscopio invertido </li><li> Las jeringas se colocan en las bombas de jeringa de alta precisión. </li><li> El sustrato, como por ejemplo una lámina de vidrio, se inserta en un soporte de fabricación casera que se fija a la platina del microscopio. </li><li> El paralelismo de la mesa de la impresora multifunción y el soporte se ajusta mediante un par de goniómetros mediante la observación de los anillos de Newton (franjas de interferencia) que aparecen cuando el MFP se pone en contacto con el sustrato. El punto de contacto y la frecuencia de los anillos de servir como indicador de la inclinación. Cuando el equipo esté alineado con la superficie, un anillo de interferencia de una sola se extiende sobre toda la superficie. Esta medida también sirve para calibrar la separación entre el MFP y el sustrato. </li><li> La brecha entre la impresora multifunción y el soporte es fundamental para los procesos de modelado de superficie. Debido a que el sustrato se procesa mediante la exploración por debajo de la impresora multifunción, la alineación horizontal tiene que ser ajustada con precisión micrométrica y se logra utilizando un soporte de tres puntos formado por tres tornillos micrométricos. </li></ol> 4. El funcionamiento de la impresora multifunción <ol><li> La dispensación es controlado a través del software LabVIEW. La operación del dispositivo se visualiza a simple vista y con una cámara CCD. La inyección: relación de aspiración varía 01:03-1:10, en función de la difusividad del reactivo con el buffer de los alrededores y el patrón de flujo deseado geométricas. </li><li> Para comprobar el correcto funcionamiento de la jeringa de aspiración y la presencia de burbujas, primero se inyecta el líquido con la jeringa de aspiración antes de iniciar la aspiración adecuada.</li><li> Comienzo de la inyección de líquido y el flujo de monitor y el confinamiento de cuentas o de tinte fluorescente marcador. </li><li> Utilice la sonda para la aplicación particular, es decir, escanear a través de la superficie para el procesamiento de la deposición, el grabado o la tinción de la superficie o las células. </li></ol>

<ol>
	<li>Juncker, D., Schmid, H., Delamarche, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature Materials. 4 (8), 622-622 (2005).</li></ol>

La sonda de microfluidos (MFP) es muy versátil, ya que es (i) móvil, (ii) adaptable para su uso con diferentes tipos de reactivos y el sustrato y se puede (iii) ser manejados en grandes áreas. Burbujas no deseado puede llevar a la interrupción del flujo Para evitar burbujas, todos los componentes tienen que ser llenado con líquidos antes de su montaje. La brecha entre la sonda y la superficie es sólo de unos pocos micrómetros, sin embargo, la mesa es de varios cientos de micrómetros de ancho, y las distancias en el rango de centímetros escaneados. Por consiguiente, tanto la horizontalidad de la superficie de escaneado y el paralelismo entre la mesa multifunción y el soporte que sea necesario ajustar con mucho cuidado. Finalmente, la relación entre la aspiración y la inyección tiene que ser lo suficientemente grande como para capturar todos los reactivos inyectados en el espacio entre el MFP y el sustrato. El MFP se puede utilizar para modelar superficies con las proteínas en condiciones suaves, para procesar los tejidos o células individuo inmerso en tampones fisiológicos, o de los patrones de grabar en una superficie.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>microfluidic connectors</td>
<td>&nbsp;</td>
<td>Upchurch Scientific</td>
<td>Micro- and Nano-tight fittings and sleeves</td>
<td>&nbsp;</td>
<tr>
<td>2-component manual dispenser</td>
<td>&nbsp;</td>
<td>Conprotec Inc.</td>
<td>DM400</td>
<td>To dispense and mix PDMS mixture</td>
</tr>
<tr>
<td>LabVIEW</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>Version 8.0</td>
<td>&nbsp;</td>
<tr>
<td>Mechanical Convection Oven</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>1330FM</td>
<td>&nbsp;</td>
<tr>
<td>Glass syringes</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Capillary tubing</td>
<td>&nbsp;</td>
<td>Polymicro Technologies</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plasma Chamber</td>
<td>&nbsp;</td>
<td>Tegal Corporation</td>
<td>Plasmaline 415</td>
<td>&nbsp;</td>
<tr>
<td>Inverted Microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>TE2000-E</td>
<td>&nbsp;</td>
<tr>
<td>Syringe pumps</td>
<td>&nbsp;</td>
<td>Cetoni</td>
<td>neMESYS</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184</td>
<td>&nbsp;</td>
<td>Ellsworth Adhesives</td>
<td>184 Sil Elast Kit</td>
<td>&nbsp;</td>
<tr>
<td>Camera</td>
<td>&nbsp;</td>
<td>Photometrics</td>
<td>QuantEM 512SC</td>
<td>&nbsp;</td>
<tr>
<td>Microscope stage</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microfluidic probe holder goniometers</td>
<td>&nbsp;</td>
<td>Melles-Griot</td>
<td>07GON504</td>
<td>&nbsp;</td>
<tr>
<td>Linear stage</td>
<td>&nbsp;</td>
<td>Applied Scientific Instrumentation</td>
<td>LS-50</td>
<td>For z-control of the MFP</td>
</tr>
<tr>
<td>Manual linear stage</td>
<td>&nbsp;</td>
<td>Newport</td>
<td>443-4 Series</td>
<td>For x- and y- axis control of the MFP</td>
</tr>
<tr>
<td>Microscope stage</td>
<td>&nbsp;</td>
<td>Applied Scientific Instrumentation</td>
<td>PZ-2000</td>
<td>With x-, y- and z- control</td>
</tr>		</tbody>
		</table>
		</div>

the microfluidic probe: operation and use for localized surface processing

Dispositivos de microfluidos permiten los ensayos que se realizaron con cantidades de muestra y se han utilizado recientemente para controlar el microambiente de las células. Microfluídica se asocia comúnmente con microcanales cerrados que limitan su uso a las muestras que pueden ser introducidas y cultivadas en el caso de las células, dentro de un volumen reducido. Por otro lado, micropipetting sistema se han utilizado para la perfusión local y las superficies de las células, sobre todo con push-pull los casos en los actos de una pipeta como fuente y el otro como lavabo, pero el confinamiento del flujo es difícil en tres dimensiones. Además, las pipetas son frágiles y difíciles de colocar y por lo tanto se utilizan en la configuración estática. La sonda de microfluidos (MFP) evita las limitaciones impuestas por la construcción de canales de microfluidos cerrado y en lugar de encerrar la muestra en el sistema de microfluidos, el flujo de microfluidos pueden ser entregados directamente en la muestra, y explora a través de la muestra, utilizando la impresora multifunción. . La inyección y las aberturas de aspiración se encuentran dentro de unas cuantas decenas de micrómetros de unos a otros para que un microjet se inyecta en el espacio es limitado por las fuerzas hidrodinámicas del líquido que rodea por completo y aspirar de nuevo en la otra abertura. El microjet se pueden eliminar a través de la superficie del sustrato y proporciona una herramienta precisa para la deposición localizada / o entrega de los reactivos que pueden ser utilizados en grandes áreas de exploración de la sonda sobre la superficie. En este vídeo se presentan los microfluidos sonda 1 (MFP). Se explica en detalle cómo ensamblar el MFP, montarlo sobre un microscopio invertido, y alinearla con respecto a la superficie del sustrato y, finalmente, mostrar cómo se utilizan para procesar una superficie de sustrato sumergido en una solución tampón.

Watch this Scientific Journal Video about The Microfluidic Probe: Operation and Use for Localized Surface Processing at JoVE.com

The Microfluidic Probe: Operation and Use for Localized Surface Processing

En este video se presenta la sonda de microfluidos<sup&gt; 1</sup&gt; (MFP). Se explica en detalle cómo ensamblar el MFP, montarlo sobre un microscopio invertido, y alinearla con respecto a la superficie del sustrato y, finalmente, mostrar cómo se utilizan para procesar una superficie de sustrato sumergido en una solución tampón.

La sonda de microfluidos: Operación y Uso de tratamiento de superficies localizadas

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

the-microfluidic-probe-operation-use-for-localized-surface

Nanyang Technological University

Research

JoVE Journal

Biology

8.8K vistas.  McGill University. En este video se presenta la sonda de microfluidos 1 (MFP). Se explica en detalle cómo ensamblar el MFP, montarlo sobre un microscopio invertido, y alinearla con respecto a la superficie del sustrato y, finalmente, mostrar cómo se utilizan para procesar una superficie de sustrato sumergido en una solución tampón.

Video: The Microfluidic Probe: Operation and Use for Localized Surface Processing

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls.
The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of ~50-150 &#181;m and extends over ~15 &#181;m around the mean penetration depth, which is adjustable between 0 and 50 &#181;m. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches.
For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery.
When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.

In this video, we show a procedure for an accurate biolistic delivery of reagents into live tissue with a novel miniature gene gun. We are knocking down the expression of the axon guidance molecule Netrin in leech embryos by delivering molecules of dsRNA into the ventral body wall and ganglia of single segments.

Localizada RNAi y la expresión génica ectópico en la sanguijuela medicinal

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

Investigación

Biología

Processing the Loblolly Pine PtGen2 cDNA Microarray

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs. The array is produced in our laboratory for use by researchers studying gene expression in pine and other conifer species. PtGen2 was developed as a result of our gene discovery efforts in loblolly pine, and is comprised of sequences identified primarily from root tissues, but also from needle and stem.1,2 PtGen2 has been tested by hybridizing different Cy-dye labeled conifer target cDNAs, using both amplified and non-amplified indirect labeling methods, and also tested with a number of hybridization and washing conditions. This video focuses on the handling and processing of slides before and after pre-hybridization, as well as after hybridization, using some modifications to procedures developed previously.3,4 Also included, in text form only, are the protocols used for the generation, labeling and clean up of target cDNA s, as well as information on software used for downstream data processing. 
PtGen2 is printed with a proprietary print buffer that contains high concentrations of salt that can be difficult to remove completely. The slides are washed first in a warm SDS solution prior to pre-hybridization. After pre-hybridization, the slides are washed vigorously in several changes of water to complete removal of remaining salts. LifterSlips&trade; are then cleaned and positioned on the slides and labeled cDNA is carefully loaded onto the microarray by way of capillary action which provides for even distribution of the sample across the slide, and reduces the chance of bubble incorporation. Hybridization of targets to the array is done at 48&deg;C in high humidity conditions. After hybridization, a series of standard washes are done at 53&deg;C and room temperature for extended times. Processing PtGen2 slides using this technique reduces salt and SDS-derived artifacts often seen when the array is processed less rigorously. Hybridizing targets derived from several different conifer RNA sources, this processing protocol yielded fewer artifacts, reduced background, and provided better consistency among different experimental groups of arrays.

The cDNA microarray PtGen2 was developed for gene expression studies in loblolly pine, P. taeda, and other conifer species. Here, we show pre- and post-hybridization handling and washing techniques that can be used with this array to yield better consistency, reduced artifacts, and lower backgrounds.

Procesamiento de los Pinus taeda PtGen2 cDNA microarray

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs.  The array is produced in our laboratory for use by researchers ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

Microfluídica digitales para el procesamiento automatizado de Proteómica

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The device can be sterilized for cell culture using common methods without loss of function. The device's applicability to studying the in vitro wound healing response will be demonstrated.

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.

Fabricación y operación de una inserción de oxígeno para Adherente cultivos celulares

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

Los ensayos biofísicos investigará las propiedades mecánicas del núcleo celular Interfase: Aplicación del sustrato deformación y manipulación de Microneedle

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

Avidez detección basada en la interacción extracelular (AVEXIS) para la detección escalable de extracelular de baja afinidad interacciones ligando-receptor

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

El uso de quimiostatos en Microbiana Biología de Sistemas

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

Inyección intraductal de entrega de la droga localizada a la glándula mamaria de ratón

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

PCR en tiempo real basada en sondeos Enfoques para la medición cuantitativa de microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Dried Blood Spots - Preparing and Processing for Use in Immunoassays and in Molecular Techniques

The idea of collecting blood on a paper card and subsequently using the dried blood spots (DBS) for diagnostic purposes originated a century ago. Since then, DBS testing for decades has remained predominantly focused on the diagnosis of infectious diseases especially in resource-limited settings or the systematic screening of newborns for inherited metabolic disorders and only recently have a variety of new and innovative DBS applications begun to emerge. For many years, pre-analytical variables were only inappropriately considered in the field of DBS testing and even today, with the exception of newborn screening, the entire pre-analytical phase, which comprises the preparation and processing of DBS for their final analysis has not been standardized. Given this background, a comprehensive step-by-step protocol, which covers al the essential phases, is proposed, i.e., collection of blood; preparation of blood spots; drying of blood spots; storage and transportation of DBS; elution of DBS, and finally analyses of DBS eluates. The effectiveness of this protocol was first evaluated with 1,762 coupled serum/DBS pairs for detecting markers of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus infections on an automated analytical platform. In a second step, the protocol was utilized during a pilot study, which was conducted on active drug users in the German cities of Berlin and Essen.

The preparing and processing of dried blood spots (DBS) for their final analysis are still poorly standardized for most diagnostic applications. To overcome this shortcoming, a comprehensive step-by-step protocol is suggested and subsequently evaluated with regard to its effectiveness for detecting markers of viral infections.

Manchas de sangre seca - preparación y procesamiento para su uso en inmunoensayos y en técnicas moleculares

The idea of collecting blood on a paper card and subsequently using the dried blood spots (DBS) for diagnostic purposes originated a century ago. Since ...