Financiado por uma doação da Fundação Nacional de Ciência para MF

A OMT é uma adaptação de um sistema de corda magnética 1, destinada a incorporar a 2 simulador virtual pluma. Os protocolos a seguir irá explicar como otimizar as imagens de vídeo adquirida (Parte 1) e executar dois tipos básicos de experimentos visuo-olfativa (Parte 2 e 3). Parte 1: Aquisição de Vídeo Otimizar a imagem de vídeo é fundamental para a qualidade dos dados adquiridos. Siga estes passos para imagens mais limpas. <ol><li> Coloque uma mosca recém amarrado na arena usando um par de fórceps. </li><li> A posição da câmera firewire bordo (Fire-I 1394store.com), sob a câmara de vácuo de acrílico transparente de tal forma que a visão da câmera é diretamente para cima através da câmara, através de um tubo de vidro, para a face ventral da mosca amarrados. Certifique-se que não há revestimento IR filtro na lente do sistema de vídeo. </li><li> Para iluminar a mosca, use um arranjo circular de LEDs infravermelhos (Small Parts-Inc) posicionado logo sob os painéis de LED. Foco cada LED no corpo da mosca individualmente para uma iluminação uniforme. </li><li> Para reduzir o brilho do sistema de painel e para minimizar pistas visuais observados pela mosca, é útil para revestir todas as superfícies reflexivas dentro da visão da câmera com tinta preta fosca. Luzes da sala deve ser desligado para reduzir o brilho e luminosidade modulações detectáveis ​​pelo olho da mosca. </li><li> Inspecionar visualmente a imagem da câmara. A mosca deve ser branco brilhante contra um fundo preto. Se a imagem é desfocada a lente pode precisar de ser focado. Se a mosca é escuro, a matriz de LEDs podem ser desfocado ou insuficientemente alimentado para uma iluminação adequada. Se o fundo contém objetos brilhantes, montar um disco de papel rebanho preto um pouco acima do fly para minimizar a reflexão de fundo. </li><li> Nós adquirimos vídeo com sub-rotinas personalizadas escritas em Matlab usando a caixa de ferramentas de aquisição de imagens. </li></ol> Parte 2: A mosca encontrar a pluma de odor Um experimento simples na OMT está observando uma mosca (fome) e depois com fome localizar ativamente rastreamento uma pluma de odor alimento atrativo. Manipular o ambiente visuais influencia a capacidade da mosca robusta para controlar um odor de alimentos apetitivo. Note-se que os experimentos são normalmente executados em um formato de blocos ao acaso para minimizar qualquer preconceito, a fim experimental. Todo o hardware externo (multiplexers de gás, arena LED, aquisição de vídeo) são controladas por rotinas de software personalizado escrito em Matlab (Mathworks). <ol><li> Coloque uma mosca recém amarrado na arena usando um par de fórceps. </li><li> Antes de cada experimento, utilizando o display visual, gire a voar por várias revoluções 3 para assegurar um movimento suave em torno do eixo de guinada todo 360. Se a mosca não gira suavemente descartá-lo. Se o problema persistir, a causa provável é ímãs desalinhados. Nota: Se a distribuição de arena indo para várias trajetórias durante o julgamento de spin mostra picos significativos (ou seja, não é plana mais de 360 ​​graus), provavelmente há um viés de orientação devido aos ímãs desalinhados. </li><li> Apresentar a voar com as condições experimentais de exemplo a seguir em ordem aleatória: i) de alto contraste-padrão listrado com vapor de água que emana do porto odor; ii) de alto contraste padrão listrado com odor atraente vapor; iii) uma única faixa deslocamento vertical de 90 graus do bico de vapor d&#39;água, e iv) a 90 graus única faixa compensada com vapor de odor. Em nossa experiência, intervalos de 30 segundos funcionam bem para os experimentos onde as moscas são desafiados a localizar a fonte do odor por conta própria. Adquirimos de vídeo de 30 Hz para a duração de cada condição experimental e analisar as imagens offline usando rotinas de software personalizado escrito em Matlab (uma das mais úteis built-in funções Matlab é regionprops.m que analisa as propriedades geométricas de uma elipse. </li><li> Quando se utiliza concentrações elevadas odor, odor residual pode ser liberado a partir do porto odor após o odor é desligado. Para minimizar os efeitos que isso possa ter, é útil para girar a voar por alguns segundos com um panorama de rotação para que o sistema para expulsar qualquer odor residual. </li><li> É também benéfico para visualmente girar a voar entre os tratamentos experimentais para (i) manter as moscas ativamente engajados no experimento e (ii) manter moscas de permanecer em um ponto. Um experimento exemplo pode ter a seguinte ordem: i) vinte rodada inicial segundo diagnóstico; ii) cinco de rotação segundo intermediários; iii) vinte par segunda condição # 1; iv) cinco girar segundo intermediários; v) vinte par segunda condição # 2. </li></ol> Parte 3: A mosca permanecendo dentro da pluma de odor Um segundo experimento básico na OMT é visualmente arrastar uma mosca em uma pluma de odor, instantaneamente mudar as condições visual, e depois medir a sua capacidade de permanecer na pluma. Novamente, note que todas as experiências são feitas em forma de blocos ao acasot para minimizar qualquer preconceito, a fim experimental. <ol><li> Coloque uma mosca recém amarrado na arena usando um par de fórceps. </li><li> Visualmente girar (spin) os tempos voam várias ao redor da arena antes de cada experiência como uma verificação de diagnóstico para garantir que o animal tem a capacidade de orientar em todas as direções (ver parte 2.2). </li><li> Antes de cada condição, ligar o odor experimental (água ou vinagre) e visualmente &quot;arrastar&quot; a voar para a pluma oscilando uma pequena faixa vertical na posição da pluma. Este aproveita o comportamento poderosa faixa de fixação observada em moscas [4]. </li><li> Agora que a mosca é orientada para a pluma, remova a faixa de oscilação e apresentar as condições visuais de exemplo a seguir: i) faixas de alto contraste panorâmico com vapor de água (Fig. 1A); ii) fundo uniforme com vapor de água (Figura 1B); iii) listras de alto contraste panorâmico com odor vapor (Fig. 1C); fundo uniforme iv) com odor de vapor (Figura 1D). Aquisição de vídeo como em 2.3. Um experimento exemplo pode ter a ordem de fluxo: i) vinte rodada inicial segundo diagnóstico; ii) cinco oscilação tarja segunda pluma; iii) vinte par segunda condição # 1; iv) cinco oscilação tarja segunda pluma; v) vinte par segunda condição # 2 . </li></ol> Resultados representativos: <img src="/files/ftp_upload/old/1110_2009_1_22_1/Fig 1.png" alt="Figura 1" width="600" height="195" /> Figura 1 Parte 1 explica como otimizar as imagens de vídeo OMT. A imagem de vídeo deve ter uma visão clara da mosca iluminado sobre um fundo escuro preto. Excessivamente imagens brilhantes pode ser melhorada através da redução da interferência de fundo, reduzindo a intensidade dos LEDs IR, ou desligando as luzes da sala. Se a mosca é mal iluminado, ajustar o foco dos LEDs IR ou a intensidade da modulação dos LEDs IR pode corrigir o problema. Partes 2 e 3 descrevem experiências simples para realizar a fim de verificar o bom funcionamento de uma OMT. Em todos os casos, uma mosca deve ser visualmente &quot;forçado&quot; (via respostas optomotor) para girar várias revoluções na arena para garantir uma ampla suave e completa de movimento. Na parte 2 de uma mosca é desafiado a encontrar uma pluma de odor por conta própria. Para manter a mosca envolvidos no experimento, para reduzir o efeito da libertação odor residual, e para manter as moscas de manter um único título durante todo o experimento, é útil para visualmente girar a voar por alguns segundos entre os ensaios. Uma mosca nunca deve localizar uma pluma de vapor d&#39;água em um grau significativo, e deve sempre localizar uma pluma de odor na presença de rica panorâmica pistas visuais. Na Parte 3, uma mosca é visualmente arrastado diretamente em uma pluma de odor e desafiados a manter o seu título dentro da pluma contra uma variedade de backgrounds estacionária visual. Se a mosca é arrastado para uma pluma de vapor d&#39;água, deve rapidamente se transformar de distância. Se a mosca é arrastado para uma pluma de odor e não são suficientes pistas visuais para mediar rastreamento robusto, a mosca permanecerá na pluma.

<ol>
	<li>Bender, J. A., Dickinson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+stimulation+of+saccades+in+magnetically+tethered+Drosophila.">Visual stimulation of saccades in magnetically tethered Drosophila.</a> J Exp Biol. 209, 3170-3182 (2006).</li><li>Duistermars, B. J., Frye, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crossmodal+visual+input+for+odor+tracking+during+fly+flight.">Crossmodal visual input for odor tracking during fly flight.</a> Curr Biol. 18, 270-275 (2008).</li><li>Reiser, M. B., Dickinson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modular+display+system+for+insect+behavioral+neuroscience.">A modular display system for insect behavioral neuroscience.</a> J Neurosci Methods. 167, 127-139 (2008).</li><li>G&#246;tz, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Course-control,+metabolism+and+wing+interference+during+ultralong+tethered+flight+in+Drosophila+melanogaster.">Course-control, metabolism and wing interference during ultralong tethered flight in Drosophila melanogaster.</a> J. Exp. Biol. 128, 35-46 (1987).</li></ol>

A aquisição das imagens para este sistema utiliza uma câmera barata placa firewire e software escrito em Matlab. Imagens claras são cruciais para a obtenção de traços precisos dados. As estratégias descritas acima são úteis para a limpeza de imagens neste sistema. Procedimentos outro vídeo de monitoramento para este sistema foram descritas 1. Além disso, este sistema pode ser facilmente modificado para incluir rastreamento em tempo real de vídeo. Os protocolos experimentais exemplo aqui descrito ...

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<td>Firewire camera</td>
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visually mediated odor tracking during flight in drosophila

Insetos voadores usar pistas visuais para estabilizar a sua posição em um fluxo de vento. Muitos animais adicionalmente faixa odores transportados pelo vento. Como tal estabilização, visual de rastreamento diretamente contra o vento auxilia no acompanhamento odor. Mas sinais olfativos influenciam diretamente o comportamento acompanhamento visual de forma independente a partir de pistas de vento? Além disso, os recentes avanços na genética molecular e olfativos neurofisiologia têm motivado novas análises quantitativas de comportamento para avaliar a influência do comportamento de (por exemplo) geneticamente inativação específica circuitos ativação olfativo. Nós modificamos um sistema de corda magnética originalmente concebido para experimentos visão, equipando a arena com estreitas laminar plumas odor de fluxo. Aqui nos concentramos em experimentos que podem ser realizadas após uma mosca é preso e é capaz de navegar na arena magnético. Nós mostramos como adquirir imagens de vídeo otimizado para medir o ângulo do corpo, como juiz de rastreamento odor estável, e nós ilustrar dois experimentos para analisar a influência de pistas visuais sobre rastreamento odor.

Watch this Scientific Journal Video about Visually Mediated Odor Tracking During Flight in Drosophila at JoVE.com

Visually Mediated Odor Tracking During Flight in Drosophila

Aqui nós descrevemos como otimizar a imagem de vídeo adquiridos para uma corda magnética olfativo (OMT) aparelho. Descrevemos também dois protocolos de amostragem experimental para estudar visuo-olfativa de fusão.

Visualmente Rastreamento Odor Mediated Durante o Voo em Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

visually-mediated-odor-tracking-during-flight-in-drosophila

Research

JoVE Journal

Biology

9.8K visualizações.  University of California, Los Angeles. Aqui nós descrevemos como otimizar a imagem de vídeo adquiridos para uma corda magnética olfativo (OMT) aparelho. Descrevemos também dois protocolos de amostragem experimental para estudar visuo-olfativa de fusão.

Vídeo: Visually Mediated Odor Tracking During Flight in Drosophila

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

De alta resolução de Medição do Comportamento Odor-Driven em larvas de Drosophila

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Biologia

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Also, the recent deluge of research on the neurophysiology and neurobehavioral genetics of olfaction in Drosophila has motivated ever more technically sophisticated and quantitative behavioral assays. Here, we modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. A fly is glued to a small steel pin and suspended in a magnetic field that enables it to yaw freely. Small diameter food odor plumes are directed downward over the fly s head, eliciting stable tracking by a hungry fly. Here we focus on the critical mechanics of tethering, aligning the magnets, devising the odor plume, and confirming stable odor tracking.

Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.

Um sistema de acoplamento magnético para Investigar controle de vôo visual e olfativo Mediated em Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

De alta resolução de vídeo de Rastreamento Locomotion em Adult Drosophila melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) 4,5,6 in Drosophila motor neurons using the GAL4-UAS system 7, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited.

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.

Channelrhodopsin2 Estimulação Mediated de Potenciais Synaptic em Drosophila Junções Neuromusculares

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentivirus mediada Durante Knockdown Ex Vivo Eritropoese de Células-Tronco Hematopoéticas

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Análise Genética rápida de epitelial-mesenquimal sinalização durante a regeneração do cabelo

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Rastreamento de celular utilizando proteínas Photoconvertible Durante o Desenvolvimento Zebrafish

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Imagens ao vivo de Apuramento celular por apoptose durante<em&gt; Drosophila</em&gt; Embriogênese

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

Um método melhorado para rápido e preciso de medição de desempenho em vôo<em&gt; Drosophila</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Uma interface de usuário gráfica para rastreamento assistido por software da concentração de proteínas em protrusões celulares dinâmicas

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...