1. La preparación de los lípidos <ol><li> A fin de vincular las proteínas de la hoja superior de una bicapa, usamos Ni 2 + lípidos quelantes, que anclan las proteínas recombinantes con poli-histidina etiquetas. Ciertos tipos de células pueden adherirse de forma inespecífica a Ni 2 + con recubrimiento bicapa, pero las células T en general, no, por lo que este método óptimo para el estudio de la activación de células T. </li><li> La solución bicapa lipídica se realiza mediante la preparación de una solución de 20% de Ni 2 + quelante de lípidos y el 80% de fosfatidilcolina en la cuantía apropiada en un tubo de vidrio con 2.1 ml de cloroformo-metanol. <ol><li> Se evapora el solvente bajo una corriente de gas nitrógeno en un ambiente cálido (30-37 ° C) baño de agua. Esto suele tardar unos 15 minutos. </li><li> Eliminar cualquier disolvente que queda en alto vacío, utilizando un liofilizador durante 90 minutos. </li></ol></li><li> Disolver los lípidos en el 2% de N-octil-glucósido detergente en Tris-buffer salina, lo que crea una solución de detergente mezclado / fosfolípido micelas. La concentración de fosfolípidos final debe ser de 0,4 mm. Para formar liposomas, diálisis de esta solución con tres cambios de Tris-salina para eliminar los liposomas detergente y forma. Por lo general, trabajan con volúmenes del orden de 1 ml con 6 mm de diámetro Spectra / Por # 2 tubos con un corte alrededor de 10 kDa. Tenemos el cuidado de excluir cualquier burbuja de aire cuando s de sujeción del tubo. Por lo general estériles filtro de esta solución y hacer la diálisis en condiciones de limpieza, la manipulación que utiliza el 70% de etanol guantes esterilizados. </li><li> Esta solución debe ser transparente. Se almacena en atmósfera de argón para evitar la oxidación. Si la solución está turbia, y luego de varias paredes vesículas se han generado, y tendrá que empezar de nuevo. </li></ol> 2. Determinar la cantidad de ICAM-1 y MHC se depositan en la bicapa plana <ol><li> Con el fin de preparar el experimento, primero es necesario determinar la cantidad de ICAM-1 y MHC se depositan en una superficie dada de Ni 2 + quelante bicapa en una determinada concentración de proteína soluble. Para ello, bicapas depósito el 5 de cuentas de vidrio micrómetro de diámetro, se incuban las perlas de vidrio con soluciones de proteínas en las condiciones que se aproximan a los de las células de flujo utilizado para la imagen y leer la densidad de las proteínas mediante un ensayo de flujo immunofluorometric. </li><li> Anticuerpos FITC marcado con un número conocido de fluoresceína por molécula se utilizan para detectar las proteínas unidas a la superficie. Fluoresceína cuentas estándar se utiliza para calibrar el flujo de microfluorimeter. </li><li> Vamos a establecer las condiciones que se aproximan a los de células presentadoras de antígenos con 200 molecules/μm2 de ICAM-1 y 0.2-20 molecules/μm2 de I-Ek-MCC91-103 complejos. </li></ol> 3. Limpieza de los cubreobjetos de vidrio para la formación de bicapas planas <ol><li> Bicapas planas se forman espontáneamente cuando los liposomas se fusionan para vidrio, pero sólo si el cristal se limpian adecuadamente. </li><li> Cuando se trabaja con Piraña, que use ropa de protección completa, incluyendo un delantal y guantes de ácido fuerte, resistente a los ácidos. Con cuidado, separar los residuos de los desechos orgánicos y deshágase de ellos apropiadamente. </li><li> Para preparar la solución Piranha ácido, agregar 75 ml de ácido sulfúrico concentrado a un vaso de seco, y añadir con cuidado 25 ml de peróxido de hidrógeno al 30%. La solución se calienta rápidamente a más de 100 ° C. </li><li> Sumerja el cubreobjetos secos en la solución durante 15 minutos. Eliminar de la solución y enjuague con agua purificada durante un minuto por cada lado. Seque el cubreobjetos con una fuente de vacío para drenar el agua purificada. Deje que la solución Piranha de enfriar y agregar al contenedor de residuos Piranha, que se queda con la tapa suelta, bien marcado en la campana extractora de humos. Cuando todos los liposomas, cubreobjetos, y las proteínas están preparados, la sinapsis inmunológica está listo para ser formado. </li></ol> 4. La formación de las bicapas <ol><li> Para formar las bicapas, nuestro laboratorio utiliza Bioptechs FCSII cámaras, que tienen la ventaja de calefacción integrado. El sistema FCSII se encuentra en una base de acero inoxidable que se fija en conjunto de un colector de microfluidos con una junta de 0,5 mm superior, un tobogán microaqueduct recubiertas con óxido de estaño e indio para la calefacción en una superficie, con ranura de fluidos en el otro lado, libre de polvo 0,25 mm junta que define la altura de la cámara de flujo y la limpieza Piranha 40 mm cubreobjetos ronda en la que se forma la bicapa. </li><li> Mientras que la hoja de la cubierta, sobre la cual se forma la doble capa, tiene que ser altamente hidrofílico y limpia, la diapositiva microacqueduct realmente funciona mejor si es más hidrofóbico. Haciendo que el hidrofóbicas microaqueduct más se logra mediante el tratamiento con 1% de HSA durante 60 minutos a temperatura ambiente, luego lavar con agua pura y el secado por completo después de este proceso de acondicionamiento, así como entre los usos. La capa de proteínas desnaturalizadas que dejan este procedimiento permite una l de suspensión de liposomas para formar una ronda, hemispherical gota que es&gt; 0,25 mm de alto. </li><li> Para montar la celda de flujo y la forma bicapas planas, coloque el colector, con los tubos dirigidos hacia arriba, sobre una superficie plana. Luego, coloque el 0. 5 mm junta con agujeros colocados en los tubos de fluidos, asegurándose de que está sentado plano. Con cuidado, coloque el lado microaqueduct en la junta con los canales de fluidos hacia arriba, y asegúrese de que se asiente plana sin aplicar ninguna presión a la baja, hasta comprobar que los orificios de la diapositiva se alinean con los tubos de microfluidos. Coloque el polvo 0,25 mm junta con un corte rectangular en la cima de la diapositiva microaqueduct para que el canal está alineada con los canales fluidos y no hay espacios de aire mayor. </li><li> Coloque un máximo de 5 gotas x 1 l de suspensión de liposomas en la superficie de la diapositiva microaqueduct en un patrón tal que la distancia de centro a centro entre cada gota es de 2,5 mm. El 5 gotas de liposomas pueden tener diferentes composiciones, pero en este caso sólo se forman dos capas dobles, uno con no Ni 2 + + lípidos y un quelante con un 10% de Ni 2 + + lípidos quelantes. </li><li> Una vez que las gotas están en su lugar, se coloca cuidadosamente la tapa de deslizamiento sobre la superficie en un solo movimiento. Abrazadera en la base lo más rápidamente posible. Las gotas de liposomas se pondrá en contacto con la hoja de la cubierta. Este contacto no se deben romper, ya que las bicapas probablemente ya han formado, cualquier interrupción puede resultar en bicapas defectuoso. </li><li> Es importante tener en cuenta para marcar las posiciones de las capas dobles con manchas de tinta algunos pequeños en la diapositiva microacqueduct para ayudar en la colocación del cubreobjetos en el microscopio. Espere 20 minutos para asegurarse de que el sistema se haya equilibrado. La transferencia de todas las soluciones con una jeringa de 20 ml conectada a unos 20 cm de tubo flexible y una llave de 3 vías. Conectar otro puerto de la llave de paso a la celda de flujo en un tramo corto de tubería para reducir al mínimo el volumen muerto entre la llave y la célula de flujo. Llene la jeringa con solución salina tamponada con HEPES que contenía 2 mM de MgCl2, 1 mM CaCl 2, 5 mM de glucosa y 1% de albúmina sérica humana. </li><li> El primer tubo de la llave de paso y para que no haya burbujas de aire. Conectar el flujo de las células y empujar a través de 5 ml en un solo movimiento mientras se ve para asegurarse de que no queden burbujas de aire pasar por encima de la bicapa. Ahora, usted tendrá su bicapas. </li></ol> Este vídeo es compatible con el artículo &quot;Hunter Gatherer y en la espalda: las sinapsis inmunológica y Kinapses como variaciones sobre el tema de la locomoción ameboide&quot; por <a href="http://arjournals.annualreviews.org/action/doSearch?action=runSearch&amp;type=advanced&amp;result=true&amp;prevSearch=%2Bauthorsfield%3A%28Dustin%2C+Michael+L.%29" target="_blank">Michael L. Dustin</a> , <a href="http://arjournals.annualreviews.org/doi/abs/10.1146/annurev.cellbio.24.110707.175226">Informe Anual de Biología Celular y del Desarrollo</a> , Volumen 24, 2008.

supported planar bilayers for the formation of study of immunological synapses and kinapse

Apoyado bicapas planas son potentes herramientas que pueden ser utilizados para modelar las interacciones moleculares en una sinapsis inmunológica. Para imitar la interacción entre los linfocitos y las células presentadoras de antígenos, se utiliza Ni2 + quelante de los lípidos para anclar la adhesión celular recombinante y proteínas MHC de la hoja superior de una bicapa con poli-histidina etiquetas. Bicapas planas son generados por la preparación de los lípidos, el tratamiento de las superficies de vidrio en la doble capa se forma, y ​​luego la formación de la bicapa en una cámara especial que contiene una celda de flujo, donde los linfocitos se añadirán. Entonces, bicapas están acusados ​​de Ni y su marcada con proteínas recombinantes se agregan. Finalmente, los linfocitos se inyectan en la celda de flujo y microscopía TIRF se puede utilizar para la formación de imágenes sinapsis y los mecanismos que controlan la locomoción de las células T, los sitios de la clasificación de los receptores, y los sitios de la degradación del receptor.

Watch this Scientific Journal Video about Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse at JoVE.com

Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Apoyado bicapas planas son potentes herramientas que pueden ser utilizados para modelar las interacciones moleculares en una sinapsis inmunológica. Aquí, se muestra los métodos para el anclaje de las proteínas de adhesión celular sabe que modulan la formación de sinapsis en el folleto superior de la bilyer lípidos y visualizar la formación de sinapsis mediante microscopía TIRF.

El apoyo de bicapa plana para la Formación de Estudio de la sinapsis inmunológica y Kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions ...

supported-planar-bilayers-for-formation-study-immunological-synapses

Research

JoVE Journal

Biology

12.5K vistas.  New York University - NYU. Apoyado bicapas planas son potentes herramientas que pueden ser utilizados para modelar las interacciones moleculares en una sinapsis inmunológica. Aquí, se muestra los métodos para el anclaje de las proteínas de adhesión celular sabe que modulan la formación de sinapsis en el folleto superior de la bilyer lípidos y visualizar la formación de sinapsis mediante microscopía TIRF.

Video: Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Prueba de sensibilidad visual a la velocidad y dirección del movimiento en lagartos

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Investigación

Biología

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Los estímulos generados por ordenador Modelo animal

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Investigating the Immunological Mechanisms Underlying Organ Transplant Rejection

La investigación de los mecanismos inmunológicos subyacentes rechazo de trasplante de órganos

Preparation of Artificial Bilayers for Electrophysiology Experiments

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can be used for many different studies, such as the characterization of membrane-active peptides, the reconstitution of ion channels or investigations on how changes in lipid bilayer properties alter the function of bilayer-spanning channels.  Here, we show how to form a planar bilayer and how to isolate small patches from the bilayer, and in a second video  will also demonstrate a procedure for using gramicidin channels to determine changes in lipid bilayer elastic properties.   We also demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Preparación de la bicapa artificial para experimentos de electrofisiología

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

Aislamiento y purificación de Drosophila Clasificación de las neuronas periféricas por bolas magnéticas

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself de dispositivos para la recuperación de las muestras criopreservadas de caída accidental en los tanques de almacenamiento criogénicos

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Introduction to Solid Supported Membrane Based Electrophysiology

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.
After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.
The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.
This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.

Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.

Introducción a Solid membrana compatibles Basada Electrofisiología

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

Direct Imaging de calcio ER con Targeted-esterasa inducida colorante de carga (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Ensayo de formación de tubos de células endoteliales para la<em&gt; In Vitro</em&gt; Estudio de la angiogénesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

De alto rendimiento de imagen Análisis de esferoides tumorales: una aplicación de software fácil de usar para medir el tamaño de esferoides forma automática y precisa

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...