1. Préparer les lipides <ol><li> Afin de lier les protéines à la notice supérieure d&#39;un bicouche, nous utilisons Ni 2 + lipides chélateurs, qui ancrent les protéines recombinantes avec des poly-histidine balises. Certains types de cellules peuvent adhérer de manière non spécifique à Ni 2 + revêtement bicouches, mais les cellules T ne sont généralement pas, ce qui rend cette méthode optimale pour l&#39;étude de l&#39;activation des cellules T. </li><li> La solution bicouche lipidique est faite par la préparation d&#39;une solution de 20% Ni 2 + lipides chélateurs et 80% en phosphatidylcholine les montants appropriés dans un tube en verre avec 1-2 ml de chloroforme-méthanol. <ol><li> Evaporer le solvant sous un courant d&#39;azote gazeux dans un cadre chaleureux (30-37 ° C) bain-marie. Cela prend habituellement environ 15 minutes. </li><li> Retirez tout solvant restant sous vide poussé, en utilisant un lyophilisateur pendant 90 minutes. </li></ol></li><li> Dissoudre les lipides dans les 2% de N-octyl-glucoside détergent dans Tris-buffer saline, ce qui crée une solution de détergent mélangé / phospholipides micelles. La concentration de phospholipides final devrait être de 0,4 mM. Pour former des liposomes, dialyser cette solution contre 3 changements de Tris-solution saline pour éliminer les liposomes de détergent et de forme. Nous travaillons habituellement avec des volumes de l&#39;ordre de 1 ml avec 6 mm de diamètre Spectra / Por # 2 avec un tube coupé environ 10 kDa. Nous prenons soin d&#39;exclure toute bulle d&#39;air s lors du serrage de la tuyauterie. Nous généralement stériles filtre cette solution et ne la dialyse dans des conditions propres, sa manipulation avec des gants stérilisés 70% d&#39;éthanol. </li><li> Cette solution doit être limpide. Il est stocké sous gaz argon pour éviter l&#39;oxydation. Si la solution est trouble, puis multi-parois des vésicules ont été générés, et vous devrez recommencer. </li></ol> 2. Déterminer combien d&#39;ICAM-1 et CMH sont déposés sur la bicouche planaire <ol><li> Afin de mettre en place l&#39;expérience, il faut d&#39;abord déterminer combien de ICAM-1 et CMH sont déposés sur une surface donnée de Ni 2 + chélateur bicouche à une concentration donnée de protéines solubles. Pour ce faire, bicouches dépôt le 5 micromètres de diamètre des perles de verre, incuber les perles de verre avec des solutions de protéines dans des conditions qui se rapprochent de celles dans les cellules de flux utilisés pour l&#39;imagerie, et lire la densité de protéine liée par un dosage de débit immunofluorometric. </li><li> Anticorps marqués au FITC avec un nombre connu de fluorescéine par molécule sont utilisés pour détecter les protéines de surface lié. Fluorescéine perles standard sont utilisées pour calibrer les microfluorimeter débit. </li><li> Nous allons créer les conditions qui se rapprochent de ceux de cellules présentatrices d&#39;antigène avec 200 molecules/μm2 d&#39;ICAM-1 et 0,2 à 20 molecules/μm2 de I-Ek-MCC91-103 complexes. </li></ol> 3. Nettoyage de la lamelles de verre pour former bicouches planaires <ol><li> Bicouches planaires se forment spontanément lorsque les liposomes fusionnent pour le verre, mais seulement si le verre est correctement nettoyé. </li><li> Lorsque vous travaillez avec Piranha, nous portons des vêtements protecteurs complets, y compris un tablier acide et lourds, l&#39;acide gantelets résistants. Soigneusement à trier les déchets à partir des déchets organiques et éliminer correctement. </li><li> Pour préparer la solution Piranha acide, ajoutez 75 ml d&#39;acide sulfurique concentré dans un bécher sec et ajouter prudemment 25 ml de peroxyde d&#39;hydrogène à 30%. La solution se réchauffe rapidement jusqu&#39;à plus de 100 ° C. </li><li> Plonger les lamelles sec dans la solution pendant 15 minutes. Retirer de la solution et rincer à l&#39;eau purifiée pour une minute de chaque côté. Sécher les lamelles à l&#39;aide d&#39;une source de vide pour évacuer l&#39;eau purifiée. Laisser la solution Piranha refroidir et ajouter au conteneur de déchets Piranha, qui se retrouve avec le bouchon lâche, bien marqué dans la hotte. Lorsque toutes les liposomes, des lamelles, et les protéines sont préparés, la synapse immunologique est prêt à être formé. </li></ol> 4. Former les bicouches <ol><li> Pour former les bicouches, notre laboratoire utilise Bioptechs FCSII chambres, qui ont les avantages de chauffage intégré. Le système FCSII est sur une base en acier inoxydable qui serre ainsi un collecteur microfluidique avec un joint de 0,5 mm en haut, une diapositive microaqueduct recouvert d&#39;oxyde d&#39;indium-étain pour le chauffage sur une surface, avec rainure fluidiques de l&#39;autre côté, un abri de la poussière de 0,25 mm joint qui définit la hauteur de la chambre d&#39;écoulement et le Piranha nettoyé 40 mm rond lamelle sur laquelle la bicouche est formée. </li><li> Alors que la lamelle, sur laquelle la bicouche est formée, a besoin d&#39;être fortement hydrophile et propre, la diapositive microacqueduct fonctionne mieux si elle est plus hydrophobe. Faire le plus hydrophobe microaqueduct est réalisée par traitement avec HSA 1% pendant 60 minutes à température ambiante, puis lavage à l&#39;eau pure et sécher complètement après ce processus de conditionnement, ainsi que entre les utilisations. Le revêtement de protéines dénaturées laissés par cette procédure permet à 1 pl de suspension de liposomes pour former un rond, hemisphericachute de L qui est&gt; 0,25 mm de haut. </li><li> Pour assembler la cellule de flux et de la forme bicouches planaires, placez le collecteur, avec les tubes dirigés vers le haut, sur une surface plane. Ensuite, placer le 0. 5 mm avec joint trous positionnés sur les tubes fluidique, en s&#39;assurant qu&#39;il est assis à plat. Placez délicatement le côté microaqueduct sur le joint avec les canaux fluidiques vers le haut et assurez-vous qu&#39;il sièges plats sans appliquer aucune pression à la baisse, jusqu&#39;à vérifier que les trous dans la lame d&#39;aligner les tubes microfluidique. Poser la poussière 0,25 mm avec un joint de taille rectangulaire en tête de la glissière microaqueduct sorte que le canal est aligné avec les canaux fluidiques et il n&#39;ya pas des espaces d&#39;air important. </li><li> Placez jusqu&#39;à 5 gouttes x 1 pi de suspension de liposomes à la surface de la lame microaqueduct dans un modèle tel que la distance de centre à centre entre chaque goutte est de 2,5 mm. Le 5 gouttes liposomes peuvent avoir des compositions différentes, mais dans ce cas nous ne pourrons former deux bicouches, l&#39;un sans Ni 2 + + lipides chélateur et un avec 10% de Ni 2 + + lipides chélateurs. </li><li> Une fois ces gouttes sont en place, placer soigneusement la lamelle sur la surface en un seul mouvement. Pince sur la base aussi vite que possible. Les gouttes de liposome devrait prendre contact avec la lamelle. Ce contact ne doit pas être rompu, puisque les bicouches ont probablement déjà formé, toute perturbation peut entraîner des bicouches défectueuses. </li><li> Il est important de se souvenir pour marquer les positions des bicouches avec quelques taches d&#39;encre sur les petites diapositives microacqueduct d&#39;aide au positionnement de la lamelle sur le microscope. Attendre 20 minutes pour s&#39;assurer que le système a équilibré. Transférer toutes les solutions en utilisant une seringue de 20 ml connecté à environ 20 cm de tube flexible et un robinet à 3 voies. Branchez un autre port du robinet à la cellule de flux par une courte longueur de tuyau pour minimiser le volume mort entre le robinet et la cellule de flux. Remplir la seringue avec une solution saline tamponnée HEPES contenant 2 mM de MgCl2, 1 mM CaCl 2, 5 mM de glucose et 1% albumine sérique humaine. </li><li> Premier de la tuyauterie et le robinet donc il n&#39;ya pas de bulles d&#39;air. Connectez-le à des cellules d&#39;écoulement et de pousser à travers 5 ml dans un mouvement tout en regardant pour s&#39;assurer qu&#39;aucune bulle d&#39;air passer au-dessus de la bicouche. Maintenant, vous aurez votre bicouches. </li></ol> Cet article soutient la vidéo &quot;Hunter Gatherer et Retour: Synapses immunologiques et Kinapses comme des variations sur le thème de la locomotion Bouldeur» par <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">Revue annuelle de biologie cellulaire et développementale</a> , tome 24, 2008.

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

Soutenu bicouches planaires sont des outils puissants qui peuvent être utilisés pour modéliser les interactions moléculaires dans une synapse immunologique. Pour imiter les interactions entre les lymphocytes et les cellules présentatrices d&#39;antigène, nous utilisons Ni2 +-chélateur lipides pour ancrer l&#39;adhésion cellulaire recombinante et de protéines du CMH à la notice supérieure d&#39;un bicouche avec le poly-histidine balises. Bicouches planaires sont générés par la préparation de lipides, de traiter les surfaces de verre où la bicouche vont se former, puis en formant la bicouche dans une chambre spécialisée contenant un flux de cellules où les lymphocytes sera ajouté. Ensuite, les bicouches sont facturés avec Ni et son étiquetées protéines recombinantes sont ajoutés. Enfin, les lymphocytes sont injectés dans la cellule de flux et microscopie TIRF peut être utilisé pour la formation des synapses image et les mécanismes de contrôle que la locomotion des cellules T, les sites de tri des récepteurs, et des sites de dégradation du récepteur.

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

Soutenu bicouches planaires sont des outils puissants qui peuvent être utilisés pour modéliser les interactions moléculaires dans une synapse immunologique. Ici, nous montrons des méthodes pour l&#39;ancrage des protéines d&#39;adhésion cellulaire connue pour moduler la formation des synapses au feuillet supérieur de la bilyer lipides et de visualiser la formation des synapses en microscopie TIRF.

Soutenu bicouches planaires pour la formation des synapses immunologiques Etude de et 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 vues.  New York University - NYU. Soutenu bicouches planaires sont des outils puissants qui peuvent être utilisés pour modéliser les interactions moléculaires dans une synapse immunologique. Ici, nous montrons des méthodes pour l'ancrage des protéines d'adhésion cellulaire connue pour moduler la formation des synapses au feuillet supérieur de la bilyer lipides et de visualiser la formation des synapses en microscopie TIRF.

Vidéo: 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.

Test sensibilité visuelle à la vitesse et la direction du mouvement chez les lézards

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

Recherche

Biologie

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.

Computer-Generated Stimuli modèle 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

Étudier les mécanismes de rejet immunologique sous-jacente de transplantation d&#39;organes

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.

Préparation de bicouches artificielle pour des expériences d&#39;électrophysiologie

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.

Isolement et purification de Drosophile Neurones périphériques en triant bille magnétique

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 dispositif de récupération d&#39;échantillons cryoconservés tombé accidentellement dans les réservoirs de stockage cryogéniques

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.

Introduction à la membrane solide étayée par électrophysiologie

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.

Imagerie directe du calcium ER avec Targeted-estérase colorant induit Chargement (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.

Des cellules endothéliales Tube Formation test pour la<em&gt; In Vitro</em&gt; Etude de l&#39;angiogenèse

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.

À haut débit d&#39;analyse de l&#39;image de Sphéroïdes tumorales: Une application logicielle conviviale pour mesurer la taille de Sphéroïdes automatiquement et avec précision

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