Queremos agradecer a S. Taneja-Bageshwar, Beifuss K., S. Kedroske y HC. Hsiao útil para los debates. Este trabajo fue financiado por la puesta en marcha de los fondos del Departamento TAMHSC de Medicina Molecular y Celular. El alcance compuesto y disco giratorio fueron comprados con fondos aportados por el departamento y la Oficina TAMHSC del Decano. Algunas cepas fueron proporcionados por el Centro de Genética Caenorhabditis, que es financiado por el Centro Nacional para Recursos de Investigación. pRF4 (rol-6 (su1006)) fue un regalo de fuego A..

1. Preparación de DII mancha <ol><li> Prepare una solución stock de 20 mg / ml DII (Biotium, Inc., Hayward, CA) en DMF. Dii es sensible a la luz, así que proteja DII de la luz, envolviendo en papel de aluminio. </li><li> Crear una dilución de trabajo de DII mediante la adición de 0,6 l de valores DII a 399,4 l M9 para cada población. Esto debería dar una dilución final de trabajo de 30 mg / ml en DII M9. Esto puede ser ampliado para la tinción de las poblaciones simultáneamente. Escudo de DII de la luz, envolviendo el tubo (s) en papel de aluminio. </li></ol> 2. Preparación de los nematodos <ol><li> Use una placa de 60 mm con una población de nematodos no contaminada. Lavado de los animales de la placa con una solución de 0,5% Triton X-100 en tampón M9 girando suavemente un líquido en un movimiento circular a través de la superficie de la placa para aflojar todos los animales adultos y larvas. Transferencia de lavado en un tubo estéril de 1,5 ml. </li><li> Inmediatamente de girar a los animales a 2000 rpm durante 30 segundos. Retire y deseche lo más apoyoernatant como sea posible sin afectar la masa de los animales en la parte inferior del tubo. </li><li> Para reducir el residual Triton X-100, enjuague con animales buffer M9, girar y retirar el sobrenadante. Repita este paso una vez. </li><li> Añadir 400 l de solución de trabajo de DII en M9 en el tubo y agitar brevemente para volver a suspender los animales en la solución. </li><li> Agite el tubo a 20 ° C horizontalmente durante 3 horas a 350 rpm en un ambiente protegido de la luz. Si lo desea, los animales se pueden incubar hasta 16 horas para la tinción. </li><li> Para reducir la cantidad de colorante no consolidados, la desaceleración de los animales a 2000 rpm durante 20 segundos. Retire y deseche tanto sobrenadante como sea posible sin afectar la masa de los animales. </li><li> Animales Resuspender en 400 l buffer M9 y vierta el líquido en una porción libre de bacterias de una placa de agar NGM sembrado con OP50 E. coli. Que los animales puedan recuperar por lo menos 30 minutos. Durante el tiempo de recuperación de los animales deben arrastrarse lejos del líquido y tinción de DII sobre los alimentos. Este pasoreduce la fluorescencia de fondo de la DII gratuita. </li></ol> 3. Montaje y la observación de muestras <ol><li> Derrita 4% de agar en agua usando un autoclave o un horno de microondas. </li><li> Cree separadores reutilizables, que pueden ser utilizados para asegurar un espesor uniforme de la almohadilla de agar, por capas de dos trozos de cinta de laboratorio sobre un portaobjetos de vidrio. Haga dos diapositivas espaciador total. </li><li> Organizar un portaobjetos de vidrio limpio entre dos diapositivas espaciador. Pipeta de unos 150 l (cuatro gotas) de agar fundido al 4% en el centro del portaobjetos de vidrio limpio. Cubrir rápidamente el agar fundido con un carro adicional para formar una plataforma de agar. Retire con cuidado la tapa deslizante, manteniendo la plataforma centrada en la parte superior de la diapositiva de montaje. </li><li> Pipeta de 5 L de nematodos anestesia (100 mM - 1 mM levamisol, por ejemplo) en la almohadilla. </li><li> Monte 8 a 12 animales de la anestesia y se cubre con un cubreobjetos microscopio. </li><li> Observar a los animales utilizando un microscopio confocal compuesto o equipados con al menos un obje 40x<html> ve y DsRed / TRITC (o compatible) del filtro. El máximo de excitación de fluorescencia de 549 nm es DII y de sus máximos de emisión es 565 nm para el tinte obligado (Biotium, Inc., Hayward, CA). </li></ol> 4. Resultados representante Manchas de DII de la cutícula de tipo salvaje y mutante C. elegans. La superficie cuticular contiene anillos separados por surcos circunferenciales y, en algunas etapas, crestas longitudinales llamadas alas. Cada etapa de desarrollo tiene estructuras cuticulares con composiciones distintas 2. Crestas o surcos de ambas alas y anillos con fluorescencia manchas, dependiendo de la composición de la superficie, a lo largo de las etapas de larva y adulto y permanecen visibles hasta un día después de la recuperación utilizando este método. Manchas de fondo fluorescentes a veces se observa (Figuras 2F, G), pero no de forma rutinaria (Figura 1, Figura 2A-E, H, I). Todas las imágenes fueron tomadas con disco giratorio confocal o, cuando así se indique, de campo amplio (WF) microscopio compuesto. ent &quot;&gt; <img alt="Figura 1" src="/files/ftp_upload/3362/3362fig1.jpg" /> Figura 1. DII manchas de la cutícula y el medio ambiente neuronas expuestas. L2 animal escénico. Ampliación de 630x. Piezas del mosaico de la imagen fueron capturados utilizando iVision Mac-software (Tecnologías de BioVision, Exton, PA). Las imágenes se unieron usando Adobe Photoshop CS3 (Adobe Systems, Inc., San Jose, CA). Barra de escala = 10 micras. Dii también con fluorescencia manchas amphid y fásmido neuronas sensoriales de la cabeza y la cola, respectivamente (flechas indican algunos). <img alt="Figura 2" src="/files/ftp_upload/3362/3362fig2.jpg" /> Figura 2. DII fluorescencia manchas C. elegans cutícula en todas las etapas de post-desarrollo embrionario. Ampliación de 630x. Barra de escala = 10 micras. Tinción de animales salvajes en una L1), B) L2; C) Dauer, D) L3; E) L4, y F) en fase adulta. Las crestas de las alas y anulares son teñidas con fluorescencia en los animales L1 y Dauer (A, C). DII manchas dorsales anular en los animales L2(B). Anular surcos mancha en L3 y L4 animales (D, E). Los surcos de las alas y anillos se tiñen en los animales adultos (FH). Alas están compuestas de dos, cinco, o tres crestas (en L1, Dauer, o los animales adultos, respectivamente) que corren a lo largo del animal (flechas) 16. Anillos crear crestas circunferencial alrededor del animal (puntas de flecha, F y J). La cutícula de vacuno mutante pantalla moderada defectos de organización cuticular (GH). G) Las crestas de las alas son discontinuos (WF). H) crestas supernumerarios alas se fusionan y ramificados o bifurcados (WF). Mutantes del gen del colágeno muestran alas y defectos anulares organización (IJ). I) En los animales transgénicos que sobreexpresan pRF4 (rol-6 (su1006)), los cantos de la mentira de las alas en un ángulo a la longitud del animal. J) anillos de transgénicos que sobreexpresan pRF4 animales (rol-6 (su1006)) muestra un patrón irregular. <img alt="Figura 3" src="/files/ftp_upload/3362/3362fig3.jpg" /> Figura 3. Externoestructuras morfológicas al son iluminadas por tinción DII. magnificación 630x. Las barras de escala = 10 micras. Dii también destaca otras características exteriores, incluyendo A) adulto hermafrodita vulva, B) los rayos de la cola masculina adulta y el ventilador, y C) hermafroditas cola pico (bífida en este contexto mutante). <img alt="Figura 4" src="/files/ftp_upload/3362/3362fig4.jpg" /> Cutícula la figura 4. Tarda más tiempo en la mancha con DII que las neuronas del medio ambiente expuestos. Ampliación de 630x. Barra de escala = 10 micras. Después de dos horas de tinción, amphid (no mostrado) y fásmido las neuronas sensoriales (flechas) son lo suficientemente manchado. Por el contrario, la cutícula de los animales más jóvenes es sólo parcialmente manchada en parches (cabeza de flecha). <table border="1"><tbody><tr><td> Lavar </td><td> Solución de tinción (+ DII) </td><td> Tiempo de incubación </td><td> Cutícula teñida </td></tr><tr><td> M9 + 0,5% TrIton X-100 </td><td> M9 + 0,5% Triton X-100 </td><td> 2 horas </td><td> no </td></tr><tr><td> M9 + 0,5% Triton X-100 </td><td> M9 + 0,5% Triton X-100 </td><td> 3 horas </td><td> no </td></tr><tr><td> M9 + 0,5% Triton X-100 </td><td> M9 </td><td> 2 horas </td><td> parcial </td></tr><tr><td> M9 + 0,5% Triton X-100 </td><td> M9 </td><td> 3 horas </td><td> sí </td></tr><tr><td> M9 + 0,5% Triton X-100 </td><td> H 2 0 </td><td> 2 horas </td><td> parcial </td></tr><tr><td> M9 + 0,5% Triton X-100 </td><td> H 2 0 </td><td> 3 horas </td><td> sí </td></tr></tbody></table> Tabla 1. Tinción de la cutícula en diferentes condiciones. Varias soluciones de incubación y los tiempos fueron probadas para optimizar tinción cuticular en los animales. H 2 0, agua destilada estéril. Coloración parcial indica manchas irregulares de la cutícula larval (Figura 4), a pesar de las manchas de adultos cutícula consistently.

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No tenemos nada que revelar.

El método de tinción DII que aquí se presenta permite una manera relativamente rápida y conveniente de visualizar la cutícula en C. elegans. Por reutilización y optimización de un método comúnmente utilizado para la imagen del medio ambiente expuestos neuronas sensoriales 15,17, DII puede ser utilizado para teñir con fluorescencia tanto alas y estructuras anulares (Figuras 1 y 2), así como la vulva, la cola masculina, y hermafroditas cola pico (Figura 3). Hemos encontrado que la solución de incubación y la influencia del tiempo de la capacidad de DII para teñir sistemáticamente la cutícula (Cuadro 1, Gráfico 4). El método de tinción de las neuronas DII medio ambiente expuestos con un tiempo de dos horas de incubación en el M9 con Triton X-100 15. Un lavado inicial de M9 con el agente tensioactivo Triton X-100 ayuda a eliminar la suciedad de la superficie de la cutícula y evita que los animales se apelmace. Sin embargo, la incubación de los animales tres horas en una solución de tinción en el mismo tampón que impide que el tinte de coloración de la cutícula (Tabla 1).Esto puede ser causado por los lípidos en la superficie del nematodo se quitó por el tratamiento más tiempo en la solución de detergente. La incubación de los animales en el agua con manchas de DII de la cutícula, lo que indica que la solución salina M9 no es necesario para la tinción de DII de la cutícula. A pesar de un protocolo a base de agua se recomienda neuronal tinción DII 15, nos encontramos con que los animales tratados de esta manera a menudo parecen poco saludables. Lavado de los animales brevemente en el 0,5% Triton X-100 en M9, seguido de una incubación de tres horas en solución de tinción hecho con M9, ofrece tinción coherente de las estructuras cuticulares y mantiene el bienestar de los animales. Los animales pueden ser rescatados después de la observación y mantenimiento, lo que permite dirigir los análisis. Este método es una herramienta útil que puede ser utilizado en muchos estudios, incluyendo pero no limitado a, la secreción de la cutícula y la organización, el desarrollo de las células epidérmicas, las vías heterochronic gen, y la evolución de los nematodos.

<table border="1"><tbody><tr><td> Reactivos </td><td> Sinónimos </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Dii </td><td> 1,1 &#39;-dioctadecil-3, 3,3&#39;, 3&#39;-tetramethylindocarbocyanine perclorato </td><td> Biotium, Inc. </td><td> 60010 </td><td> Archivo de dilución: 20 mg / mL de DMF dilución de trabajo: 30 mg / ml </td></tr><tr><td> DMF </td><td> Dimetilformamida </td><td> Sigma-Aldrich, Inc. </td><td> D4551 </td><td></td></tr><tr><td> Tritón X-100 </td><td> Octylphenoxypolyethoxyethanol </td><td> VWR International, LLC. </td><td> EM-9410 </td><td></td></tr><tr><td> M9 </td><td> 22mm KH 2 PO 4, 42 mm de Na 2 HPO 4, 86 mm NaCl, 1 mM MgSO 4 </td><td></td><td></td><td></td></tr><tr><td> NGM &lt;/ Td&gt; <td> Nematodo medio de crecimiento </td><td> IPM Scientific, Inc. </td><td> 11006-501 </td><td> Se pueden preparar según el protocolo de NGM agar 18 </td></tr><tr><td> Agar agar </td><td></td><td> EMD Chemicals, Inc. </td><td> 1,01614 </td><td> 4% en agua </td></tr><tr><td> Levamisol </td><td> Levamisol clorhidrato </td><td> Sigma-Aldrich, Inc. </td><td> 31742 </td><td> 100 mM - 1 mM levamisol como lo requiere </td></tr><tr><td> Portaobjetos de microscopio </td><td></td><td> VWR International, LLC. </td><td> 16005-106 </td><td></td></tr><tr><td> Gafas microscopio cubrir </td><td></td><td> VWR International, LLC. </td><td> 16004-302 </td><td></td></tr><tr><td> Compuesto alcance </td><td></td><td> Carl Zeiss, Inc. </td><td> A1m </td><td> Utilice los objetivos para satisfacer las necesidades del experimento </td></tr><tr><td> Filtro TRITC compatible u otro </td><td> Ynbsp; </td><td> Chroma Technology Corp. </td><td> 49005 </td><td> ET - DsRed (TRITC/Cy3) farfulló conjunto de filtros </td></tr></tbody></table>

visualization of caenorhabditis elegans cuticular structures using the lipophilic vital dye dii

La cutícula de la C. elegans es una estructura muy resistente que rodea el exterior del animal 1-4. La cutícula no sólo protege al animal del medio ambiente, sino que también determina la forma del cuerpo y juega un papel en la motilidad 4-6. Varias capas secretada por las células epidérmicas forman la cutícula, que incluye una capa lipídica externa 7. Crestas circunferenciales en la cutícula del llamado patrón de anillos de la longitud del animal, y están presentes en todas las etapas de desarrollo 8. Las alas son crestas longitudinales que están presentes durante las etapas específicas del desarrollo, incluyendo L1, Dauer, y adulto 2,9. Las mutaciones en los genes que afectan a la organización del colágeno cuticular puede alterar la estructura de la cutícula y la morfología corporal del animal 5,6,10,11. Mientras que imágenes cuticular mediante microscopía óptica compuesto con DIC es posible, métodos actuales que ponen de relieve las estructuras cuticulares son fluorescentesciento de expresión del transgen 12, la tinción de anticuerpos 13, y la microscopía electrónica 1. Etiquetados aglutinina de germen de trigo (WGA) también se ha utilizado para visualizar las glicoproteínas cuticular, pero está limitado en la resolución de las estructuras más finas cuticular 14. Tinción de la superficie de la cutícula con un tinte fluorescente se ha observado, pero nunca caracterizado en detalle 15. Se presenta un método para visualizar la cutícula en vivo C. elegans con el fluorescente de color rojo lipofílico DII colorante (1,1 &#39;-dioctadecil-3, 3,3&#39;, 3&#39;-tetramethylindocarbocyanine perclorato), que se utiliza comúnmente en C. elegans para visualizar las neuronas el medio ambiente expuestos. Este protocolo optimizado para la tinción de Dii es un método simple y robusto para la visualización de alta resolución de anillos fluorescentes, las alas, la vulva, la cola masculina, y hermafroditas cola aumento en C. elegans.

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Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

Se presenta un método para visualizar la cutícula en vivo<em&gt; C. elegans</em&gt; Con el fluorescente de color rojo lipofílico DII colorante (1,1 &#39;-dioctadecil-3, 3,3&#39;, 3&#39;-tetramethylindocarbocyanine perclorato), que se utiliza comúnmente en<em&gt; C. elegans</em&gt; Para visualizar las neuronas el medio ambiente expuestos. Con este protocolo optimizado, las estructuras de alas y de anular la cutícula están manchadas por el DII y observó mediante microscopía de compuesto.

La visualización de Caenorhabditis Elegans Estructuras de la cutícula uso del colorante lipofílico Vital, DII

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

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Biology

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

17.0K vistas.  Texas A&M University System Health Science Center, College of Medicine. Se presenta un método para visualizar la cutícula en vivo C. elegans Con el fluorescente de color rojo lipofílico DII colorante (1,1 '-dioctadecil-3, 3,3', 3'-tetramethylindocarbocyanine perclorato), que se utiliza comúnmente en C. elegans Para visualizar las neuronas el medio ambiente expuestos. Con este protocolo optimizado, las estructuras de alas y de anular la cutícula están manchadas por el DII y observó mediante micr...

Video: Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

Medición Caenorhabditis elegans Duración de la vida en 96 placas de microtitulación Bueno

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

Investigación

Biología

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Básico<em&gt; Caenorhabditis elegans</em&gt; Métodos de sincronización y de observación

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

El cultivo de<em&gt; Caenorhabditis elegans</em&gt; En tres dimensiones en el Laboratorio

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1&#8217;-Dioctadecy-3,3,3&#8217;,3&#8217;-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl&#8217;s blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Using a lipophilic 1,1&#39;-Dioctadecy-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature.

La perfusión como un método para la visualización vascular<em&gt; Ambystoma mexicanum</em

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

Enfoques cuantitativos para el estudio de las estructuras celulares y la morfología de los organélilos en Los cenoheman

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

Purificación y análisis de las vesículas extracelulares de Caenorhabditis elegans

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

Uso de Caenorhabditis elegans para detectar interacciones de chaperonas específicas del tejido

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

Automatización de la cuantificación agregada en Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and sequencing. The traditional proteinase K-based methods for genomic DNA extraction from C. elegans take several hours. Commercial extraction kits that effectively break open the C. elegans cuticle and extract genomic DNA are limited. An easy, faster (~15 min), and cost-efficient method of extracting C. elegans genomic DNA that works well for classroom and research applications is reported here. This DNA extraction method is optimized to use single or a few late-larval (L4) or adult nematodes as starting material for obtaining a reliable template to perform PCR. The results indicate that the DNA quality is suitable for amplifying gene targets of different sizes by PCR, permitting genotyping of single or a few animals even at dilutions to one-fiftieth of the genomic DNA from a single adult per reaction. The reported protocols can be reliably used to quickly produce DNA template from a single or a small sample of C. elegans for PCR-based applications.

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Presented here is a description of the straightforward and relatively rapid isolation of Caenorhabditis elegans genomic DNA from one or a few animals using a commercially available tissue kit. The resulting gDNA preparation is a suitable template for PCR.

Extracción a pequeña escala de ADN genómico de Caenorhabditis elegans

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and ...