Financiado por una beca de la Fundación Nacional de Ciencias de MF

La OMT es una adaptación de un sistema de sujeción magnética 1, destinada a incorporar un 2 penacho simulador virtual. Los siguientes protocolos se explica cómo optimizar las imágenes de vídeo adquirida (Parte 1) y ejecutar dos tipos de básicos viso-olfativas experimentos (Parte 2 y 3). Parte 1: Adquisición de vídeo La optimización de la imagen de vídeo es crucial para la calidad de los datos adquiridos. Siga estos pasos para imágenes más nítidas. <ol><li> Coloque una mosca recién atados en la arena con un par de pinzas. </li><li> La posición de la placa de la cámara firewire (Fuego-I 1394store.com) en la cámara de vacío de acrílico transparente de manera que la vista de la cámara directamente hacia arriba a través de la cámara, a través de un tubo de vidrio, a la cara ventral de la mosca atada. Asegúrese de que no hay capa de filtro IR en el objetivo del sistema de vídeo. </li><li> Para iluminar la marcha, utilizar una matriz circular de LEDs infrarrojos (piezas pequeñas Inc) situada justo debajo de los paneles LED. Enfoque de cada LED en el cuerpo de la mosca de forma individual para una iluminación uniforme. </li><li> Para reducir los reflejos del sistema de paneles y reducir al mínimo las señales visuales observadas por la mosca, es útil para cubrir todas las superficies reflectantes dentro de la cámara con planos de pintura color negro. Luces de la habitación debe estar apagado para reducir el resplandor y el brillo de las modulaciones detectable por el ojo de la mosca. </li><li> Inspeccione visualmente la imagen de la cámara. La mosca debe ser de color blanco brillante contra un fondo negro. Si la imagen es borrosa la lente tenga que estar concentrado. Si la marcha es oscura, la matriz de LED puede ser fuera de foco o insuficiente potencia para la iluminación adecuada. Si el fondo contiene los objetos brillantes, montar un disco de papel de rebaño negro justo por encima de la marcha para minimizar la reflexión de fondo. </li><li> Adquirimos de vídeo con las subrutinas personalizadas escritas en Matlab usando la caja de herramientas de adquisición de imágenes. </li></ol> Parte 2: La mosca de la búsqueda de la pluma de olor Un simple experimento en la OMT está cumpliendo con un hambre (hambre) y luego volar la localización de un seguimiento activo de una nube de olor a comida atractiva. Manipular el entorno influye en la capacidad visual de la mosca de la pista con firmeza un olor del alimento apetito. Tenga en cuenta que los experimentos se ejecutan normalmente en un formato de bloques al azar para minimizar el sesgo con el fin experimental. Todo el hardware externo (multiplexores gas, arena LED, la adquisición de vídeo) están controlados por las rutinas de software a medida en Matlab (Mathworks). <ol><li> Coloque una mosca recién atados en la arena con un par de pinzas. </li><li> Antes de cada experimento, utilizando la pantalla visual, haga girar la marcha de varias revoluciones 3 para asegurar un movimiento suave en todo el eje de guiñada total de 360. Si la marcha no gira con suavidad desprenderse de ella. Si el problema persiste, la causa más probable es imanes alineados. Nota: Si la distribución de la arena en dirección a varias trayectorias durante el juicio giro muestra picos significativos (es decir, no es plana en 360 grados), es posible que haya un sesgo debido a la orientación de los imanes alineados. </li><li> Presente al vuelo con las condiciones siguiente ejemplo experimental en orden aleatorio: i) de alto contraste patrón de rayas con vapor de agua que emana desde el puerto de olor, ii) de alto contraste patrón de rayas con un olor atractivo de vapor, y iii) una raya vertical único de compensación de 90 grados de la boquilla de vapor de agua, y iv) una sola banda de 90 grados de compensación con el vapor de olor. En nuestra experiencia, a intervalos de 30 segundos funcionan bien para los experimentos en moscas tienen el reto de localizar la fuente de olor por sí mismos. Adquirimos de vídeo a 30 Hz para la duración de cada condición experimental y analizar las imágenes en línea con las rutinas de software a medida en Matlab (uno de los más útiles funciones incorporadas de Matlab es regionprops.m que analiza las propiedades geométricas de una elipse. </li><li> Cuando se utilizan altas concentraciones de olores, el olor residual puede ser lanzado desde el puerto de olor después de que el olor está apagado. Para minimizar los efectos que esto puede tener es útil para hacer girar la marcha durante unos segundos con un panorama de rotación para que el sistema para eliminar cualquier mal olor residual. </li><li> También es beneficioso para rotar visualmente la marcha entre los tratamientos experimentales para (i) mantener las moscas participan activamente en el experimento y (ii) mantener a las moscas de permanecer en un solo lugar. Un experimento de ejemplo puede tener el siguiente orden: i) veinte segundos giro de diagnóstico inicial; ii) cinco segunda vuelta intermedios, iii) veinte pares segunda condición # 1; iv) cinco segundos centrifugado intermedio; v) veinte pares segunda condición # 2. </li></ol> Parte 3: La mosca de permanecer dentro de la pluma de olor Un segundo experimento básico en la OMT es arrastrar visualmente una mosca en una nube de olor, instantáneamente cambiar las condiciones visuales, y luego medir su capacidad para permanecer en la pluma. Una vez más, tenga en cuenta que todos los experimentos se hacen en una forma de bloques al azart para minimizar cualquier sesgo con el fin experimental. <ol><li> Coloque una mosca recién atados en la arena con un par de pinzas. </li><li> Visualmente girar (spin) de los tiempos vuelan varias alrededor de la arena antes de cada experimento como una verificación de diagnóstico para asegurarse de que el animal tiene la capacidad de orientarse en cualquier dirección (ver apartado 2.2). </li><li> Antes de cada estado, a su vez en el olor experimental (agua o vinagre) y visual de &quot;arrastrar&quot; la mosca en la columna oscilante por una pequeña franja vertical en la posición de la pluma. Este se aprovecha de la conducta de fijación poderosa banda observada en las moscas [4]. </li><li> Ahora que la marcha se orienta hacia la columna, eliminar la franja de oscilación y presentan las condiciones de la muestra visual de lo siguiente: i) franjas de alto contraste panorámica con vapor de agua (Fig. 1A), ii) el fondo uniforme de vapor de agua (Fig. 1B); iii) franjas de alto contraste panorámica con olor a vapor (Fig. 1C); iv) uniformes de fondo con el olor de vapor (Fig. 1D). Adquirir el vídeo como en 2.3. Un experimento de ejemplo puede tener la orden de flujo: i) veinte segundos giro inicial de diagnóstico; ii) cinco segundos banda de oscilación pluma; iii) veinte pares segunda condición # 1; iv) cinco segundos banda de oscilación pluma; v) veinte pares segunda condición # 2 . </li></ol> Los 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 cómo optimizar las imágenes de vídeo OMT. La imagen de vídeo debe tener una visión clara de la mosca iluminados sobre un fondo negro oscuro. Demasiado brillantes imágenes se pueden mejorar mediante la reducción de la interferencia de fondo, lo que reduce la intensidad de los LEDs IR, o apagando las luces del cuarto. Si la mosca no está bien iluminado, de ajustar el enfoque de los LEDs IR o la modulación de la intensidad de los LEDs IR puede solucionar el problema. Partes 2 y 3 describen experimentos sencillos para llevar a cabo para verificar el correcto funcionamiento de una OMT. En todos los casos, una mosca debe ser visualmente &quot;forzada&quot; (a través de las respuestas optomotor) para girar varias revoluciones en el campo para asegurar una amplia y fluida de movimiento. En la Parte 2 de una mosca es el reto de encontrar una pluma de olor por sí mismo. Para mantener la marcha participan en el experimento, para reducir el efecto de la liberación de olor residual, y mantener a las moscas de mantener una sola línea durante todo el experimento, es útil rotar visualmente la marcha durante unos segundos entre los ensayos. Una mosca no debe localizar una nube de vapor de agua en un grado significativo, y siempre se debe localizar una pluma de olor en la presencia de ricos señales visuales panorámicas. En la parte 3 de una mosca es visualmente arrastrar directamente en una pluma de olor y el reto de mantener su rumbo dentro de la pluma contra una variedad de fondos fijos visual. Si la mosca se arrastra en una nube de vapor de agua, rápidamente se debe rechazar. Si la mosca se arrastra en una nube de olor y hay suficientes indicaciones visuales para mediar en el seguimiento de robusta, la mosca se mantendrá en la 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>

La adquisición de las imágenes de este sistema utiliza una cámara firewire placa de bajo costo y software escrito en Matlab. Imágenes claras son cruciales para la obtención de trazas de datos precisos. Las estrategias descritas anteriormente son útiles para la limpieza de las imágenes en este sistema. Otros procedimientos de seguimiento de vídeo de este sistema se han descrito 1. Además, este sistema podría ser fácilmente modificado para incluir seguimiento en tiempo real de vídeo. Los protocolos ...

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

Los insectos voladores usar señales visuales para estabilizar su título en una corriente de viento. Muchos animales, además, la pista lleva a los olores en el viento. Por lo tanto, la estabilización visual de seguimiento directamente contra el viento ayuda en el seguimiento de olor. Pero las señales olfativas directamente influyen en el comportamiento de seguimiento visual independientemente de las señales de viento? Además, los recientes avances en genética molecular olfativa y la neurofisiología, han motivado la novela analiza el comportamiento cuantitativo para evaluar la influencia del comportamiento de (por ejemplo) genéticamente la inactivación específica de circuitos de activación olfativa. Hemos modificado un sistema de sujeción magnética originalmente ideado para experimentos de visión y dotar a la arena con plumas estrechas laminar olor del flujo. Aquí nos centramos en los experimentos que se pueden realizar después de una mosca se ata y es capaz de navegar en el campo magnético. Se muestra cómo la adquisición de imágenes de vídeo optimizado para medir el ángulo del cuerpo, ¿cómo juzgar el seguimiento de olor estable, y nos muestran dos experimentos para examinar la influencia de los estímulos visuales en el seguimiento de olor.

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

Aquí se describe cómo optimizar la imagen de vídeo adquirido por un olfato magnético de sujeción (OMT) aparato. También describe dos protocolos de la muestra experimental para el estudio visual-olfativa de fusión.

Seguimiento visual mediada olor durante el vuelo en 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 vistas.  University of California, Los Angeles. Aquí se describe cómo optimizar la imagen de vídeo adquirido por un olfato magnético de sujeción (OMT) aparato. También describe dos protocolos de la muestra experimental para el estudio visual-olfativa de fusión.

Video: 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 resolución de medición del comportamiento de olor-Driven en larvas de Drosophila

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

Investigación

Biología

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.

Un Sistema de Anclaje magnética para investigar control mediado de vuelo visual y olfativa en 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.

Vídeo de alta resolución de Seguimiento de la locomoción en adultos de 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 estimulación mediada de los potenciales sinápticos en Drosophila Las uniones 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.

Lentiviral mediada por derribo Durante Ex Vivo Eritropoyesis de las células estaminales hematopoyéticas

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.

El análisis genético rápido de epitelio-mesenquimal de señalización durante la regeneración del cabello

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.

Celular seguimiento mediante fotoconvertible proteínas durante el desarrollo de pez cebra

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.

Imágenes de Apoptotic Liquidación de células vivas durante<em&gt; Drosophila</em&gt; Embriogénesis

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.

Un método mejorado para la rápida y precisa medición de rendimiento en vuelo<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 ...

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight muscles (IFMs) of flies, specifically the dorso-longitudinal muscles (DLM), as the experimental subject to measure the atrophic phenotype brought about by different genetic causes. In this protocol, we describe how to embed fly thorax muscles for semi thin sectioning, how to obtain a good contrast between muscle and the surrounding tissue, and how to process optical microscope images for semiautomatic acquisition of quantifiable data and analysis. We describe three specific applications of the methodological pipeline. First, we show how the method can be applied to quantify muscle degeneration in a myotonic dystrophy fly model; second, measurement of muscle cross-sectional area can help to identify genes that either promote or prevent muscle atrophy and/or muscle degeneration; third, this protocol can be applied to determine whether a candidate compound is able to significantly modify a given atrophic phenotype induced by a disease-causing mutation or by an environmental trigger.

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

We report a method to quantify muscle area, which is an indirect method to determine muscle mass in Drosophila adults. We demonstrate the application of our methodology by analyzing the indirect flight muscles in a Drosophila model of Myotonic Dystrophy disease.

Determinación de zona óptica transversal muscular de los músculos de vuelo indirectos adultos de Drosophila Melanogaster

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight ...