1. Preparar os lipídios <ol><li> A fim de ligar proteínas para o folheto superior de uma bicamada, usamos Ni 2 + lipídios quelante, que escora proteínas recombinantes com poli-histidina tags. Certos tipos de células podem aderir não especificamente para bicamadas + revestido Ni 2, mas as células T, geralmente, não, tornando este método ideal para estudar a ativação de células T. </li><li> A solução bicamada lipídica é feita através da preparação de uma solução de Ni 2 + 20% de lipídios e 80 quelante fosfatidilcolina% no montante adequado a um tubo de vidro com 1-2 ml de clorofórmio-metanol. <ol><li> Evaporar o solvente sob um fluxo de gás nitrogênio em um ambiente aconchegante (30-37 ° C) em banho maria. Isso normalmente leva cerca de 15 minutos. </li><li> Remover qualquer traço do solvente sob alto vácuo, usando um liofilizador por 90 minutos. </li></ol></li><li> Dissolver os lipídios em 2% N-octil-glucoside detergente em tampão Tris-salina, o que cria uma solução de mistura detergente / fosfolipídio micelas. A concentração de fosfolipídios final deve ser 0,4 mM. Para formar os lipossomas, diálise esta solução com três mudanças de Tris-salina para remover lipossomas detergente e forma. Costumamos trabalhar com volumes da ordem de 1 ml com 6 mm de diâmetro Spectra / por # 2 tubos com um corte em torno de 10 kDa. Temos o cuidado de excluir qualquer bolha de ar quando s de fixação da tubulação. Nós normalmente estéril-filtro esta solução e fazer a diálise em condições limpas, manuseá-lo usando luvas de etanol 70% esterilizados. </li><li> Esta solução deve ser cristalina. Ela é armazenada sob gás argônio para evitar a oxidação. Se a solução estiver turva, então multi-walled vesículas foram gerados, e você terá que começar novamente. </li></ol> 2. Determinar a quantidade de ICAM-1 e MHC são depositados no bicamada planar <ol><li> , A fim de configurar o experimento, é necessário primeiro determinar o quanto ICAM-1 e MHC são depositados em uma área determinada superfície de Ni 2 + quelante bicamada em uma dada concentração de proteína solúvel. Para fazer isso, bicamadas depósito em 5 micrômetros de diâmetro contas de vidro, incubar as esferas de vidro com soluções de proteínas em condições que se aproximem daquelas nas células de fluxo usado para geração de imagens, e ler a densidade de proteínas ligadas por um fluxo de ensaio imunofluorométrico. </li><li> Anticorpos marcados com FITC-número conhecido de fluoresceína por molécula são usados ​​para detectar as proteínas de superfície-bound. Contas de fluoresceína padrão são usados ​​para calibrar o fluxo de microfluorimeter. </li><li> Vamos estabelecer as condições que se aproximem daquelas em células apresentadoras de antígenos, com 200 molecules/μm2 de ICAM-1 e 0,2-20 molecules/μm2 de I-Ek-MCC91-103 complexos. </li></ol> 3. A limpeza da lamínulas de vidro para a formação de bicamadas planar <ol><li> Bicamadas planas formam espontaneamente quando o lipossomas fusível de vidro, mas só se o copo está devidamente limpo. </li><li> Ao trabalhar com Piranha, usamos roupas de proteção total, incluindo um avental de ácido e pesado, ácido luvas resistentes. Cuidadosamente segregar os resíduos a partir de resíduos orgânicos e descartar adequadamente. </li><li> Para preparar a solução de ácido Piranha, adicione 75 ml de ácido sulfúrico concentrado a um recipiente seco e, cuidadosamente, adicionar 25 ml de peróxido de hidrogênio 30%. A solução rapidamente se aquece para mais de 100 ° C. </li><li> Mergulhe as lamelas seco na solução por 15 minutos. Remover da solução e enxaguar em água purificada por um minuto em cada lado. Seca as lamelas utilizando uma fonte de vácuo para drenar a água purificada. Permitir que a solução Piranha para esfriar, e adicionar ao recipiente de resíduos Piranha, que fica com a tampa solta, bem marcado na capela. Quando todos os lipossomas, lamínulas, e as proteínas são preparados, a sinapse imunológica está pronto para ser formado. </li></ol> 4. Formando o bicamadas <ol><li> Para formar a bicamadas, nosso laboratório utiliza Bioptechs FCSII câmaras, que têm as vantagens de aquecimento integrado. O sistema FCSII está em uma base de aço inoxidável que grampos em conjunto um coletor microfluídicos com uma junta de 0,5 mm de topo, um slide microaqueduct revestido com óxido de índio e estanho para o aquecimento em uma superfície, com sulco fluídico do outro lado, um livre de poeira 0,25 milímetros junta que define a altura da câmara de fluxo ea Piranha limpos lamela 40 mm redondo em que a bicamada é formada. </li><li> Enquanto a lamínula, em que a bicamada é formada, precisa ser altamente hidrofílico e limpo, o slide microacqueduct realmente funciona melhor se é mais hidrofóbica. Tornando o mais hidrofóbico microaqueduct é alcançada após o tratamento com HSA 1% por 60 minutos em temperatura ambiente, em seguida, lavar com água pura e secagem completamente após este processo de condicionamento, bem como entre os usos. O revestimento de proteínas desnaturadas deixadas por este procedimento permite que 1 ml de suspensão de lipossomas para formar um círculo, hemispherical gota que é&gt; 0,25 mm de altura. </li><li> Para montar a célula de fluxo e formar bicamadas planar, coloque o manifold, com os tubos direccionado para cima, em cima de uma superfície plana. Então, coloque a 0. 5 junta mm com furos posicionado sobre os tubos fluídico, certificando-se que ele esteja bem encaixada. Delicadamente, coloque o lado microaqueduct para a junta com os canais fluídicos voltada para cima, e certifique-se que assentos lisos sem aplicar qualquer pressão para baixo, até verificar que os buracos no slide se alinham com os tubos microfluídicos. Coloque o livre de poeira junta 0,25 mm com um corte retangular no topo do slide microaqueduct para que o canal está alinhado com os canais fluídicos e que não existem espaços de ar maiores. </li><li> Coloque até 5 x 1 gotas mL de suspensão de lipossomas na superfície da lâmina microaqueduct em um padrão tal que a distância de centro a centro entre cada gota é de 2,5 mm. A 5 gotas lipossomas podem ter composições diferentes, mas neste caso só vamos formar duas bicamadas, um sem Ni 2 + + lipídios quelante e um com 10% Ni 2 + + lipídios quelante. </li><li> Uma vez que estas gotas estão no lugar, coloque cuidadosamente a tampa de deslizamento sobre a superfície em um movimento. Pinça para a base o mais rápido possível. As gotas lipossomas devem fazer contato com a lamínula. Este contato não deve ser quebrado, já que o bicamadas provavelmente já formado; qualquer interrupção pode resultar em bicamadas com defeito. </li><li> É importante lembrar para marcar as posições das bicamadas com algumas pequenas manchas de tinta no slide microacqueduct para auxiliar na colocação da lamela no microscópio. Esperar 20 minutos para se certificar de que o sistema tenha equilibrada. Transferência de todas as soluções usando uma seringa de 20 ml conectada a cerca de 20 cm de tubos flexíveis e uma torneira de 3 vias. Conectar outra porta da torneira para a célula de fluxo por um curto período de tubulação para minimizar o volume morto entre a torneira ea célula de fluxo. Encha a seringa com HEPES-salina tamponada contendo 2 mM MgCl 2, 1 mM CaCl 2, 5 mM de glicose e 1% albumina sérica humana. </li><li> Primeiro-tubo e torneira para que não haja bolhas de ar. Conecte-o ao fluxo de células e empurrar a 5 ml em um movimento, enquanto observa a certeza de que nenhuma bolha de ar passar sobre a bicamada. Agora, você terá a sua bicamadas. </li></ol> Este artigo de vídeo suporta &quot;Hunter Gatherer e para trás: Sinapses imunológicas e Kinapses como variações sobre o tema da Locomotion Amoeboid&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">Revisão Anual de Biologia Celular e do Desenvolvimento</a> , Volume 24, 2008.

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

Suportados bicamadas planar são ferramentas poderosas que podem ser usados ​​para modelar as interações moleculares em uma sinapse imunológica. Para simular as interações entre linfócitos e células apresentadoras de antígenos, usamos Ni2 +-quelante lipídios para ancorar adesão celular e proteínas recombinantes MHC o folheto superior de uma bicamada com poli-histidina tags. Bicamadas planas são gerados através da preparação de lipídios, o tratamento de superfícies de vidro, onde a bicamada formará, e então formar a bicamada em uma câmara especializada contendo uma célula de fluxo onde os linfócitos serão adicionados. Então, bicamadas são acusados ​​de Ni e sua marcadas proteínas recombinantes são adicionados. Finalmente, os linfócitos são injetados na célula de fluxo e microscopia TIRF pode ser usado para formação da imagem sinapse e os mecanismos que controlam a locomoção de células T, sites de classificação receptor, e os locais de degradação 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

Suportados bicamadas planar são ferramentas poderosas que podem ser usados ​​para modelar as interações moleculares em uma sinapse imunológica. Aqui, vamos mostrar os métodos de ancoragem de proteínas de adesão celular que modula a formação de sinapses para o folheto superior do bilyer lipídico e visualize a formação de sinapses através de microscopia TIRF.

Suportados Bicamadas Planar para a Formação do Estudo da Sinapses imunológicas e 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 visualizações.  New York University - NYU. Suportados bicamadas planar são ferramentas poderosas que podem ser usados ​​para modelar as interações moleculares em uma sinapse imunológica. Aqui, vamos mostrar os métodos de ancoragem de proteínas de adesão celular que modula a formação de sinapses para o folheto superior do bilyer lipídico e visualize a formação de sinapses através de microscopia TIRF.

Vídeo: 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.

Teste de sensibilidade visual para a velocidade e direção do movimento em Lagartos

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

Biologia

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.

Gerados por computador Modelo Estímulos 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

Mecanismos imunológicos investigando a rejeição do transplante de órgãos Subjacente

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.

Preparação de Bicamadas Artificial para Experimentos Eletrofisiologia

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.

Isolamento e purificação de Drosophila Neurônios periféricos por classificação Bead Magnética

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 Dispositivo de Recuperação de amostras criopreservadas cair acidentalmente em Tanques de armazenamento criogênico

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.

Introdução à membrana apoiada sólida baseada Eletrofisiologia

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.

Imagem direta de cálcio ER com Targeted-Esterase Induzida Dye Loading (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.

Endotelial Tubo celular Formação ensaio para a<em&gt; In Vitro</em&gt; Estudo da angiogênese

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 alta capacidade Análise de Imagem de esferóides tumorais: uma aplicação de software de fácil utilização para medir o tamanho de esferóides automaticamente e com precisão

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