Name: a buoyancy-based method of determining fat levels in drosophila
Uploaded: 2016-11-01T11:15:00
Description: Watch this Scientific Journal Video about A Buoyancy-based Method of Determining Fat Levels in Drosophila at JoVE.com

K.E.H. was supported by the Training Grant in Molecular Biology NIH-T32-GM08730. This work was supported by NIH, NIDDK Grant 5K01DK095932 and AHA Award 12BGIA11930014 to T.R.

1. recoger huevos de moscas con genotipos de interés NOTA: cruces Ideal constan de 150 moscas vírgenes y al menos 75 varones. colecciones de archivo debe consistir de por lo menos 200 moscas. <ol><li> Preparar placas de uva. <ol><li> Añadir 37,5 g de agar a 1,5 L de agua destilada en un matraz de 4 L. </li><li> Autoclave mezcla de agua / agar durante 50 minutos a 121 ºC (250 ºF). </li><li> Mientras que el agua / mezcla de agar es el tratamiento en autoclave, añadir 50 g de sacarosa al jugo de uva 500 ml ...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+studying+metabolism+in+Drosophila.">Methods for studying metabolism in Drosophila.</a> Methods. 68, 105-115 (2014).</li><li>Al-Anzi, B., Zinn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+measurement+of+triglycerides+cannot+provide+an+accurate+measure+of+stored+fat+content+in+Drosophila.">Colorimetric measurement of triglycerides cannot provide an accurate measure of stored fat content in Drosophila.</a> PLoS One. 5 (8), e12353 (2010).</li><li>Thanh, N. T. 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Technol. 117, 274-280 (2015).</li><li>Shui, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+one+step+analysis+of+cellular+lipidomes+using+liquid+chromatography+coupled+with+mass+spectrometry:+application+to+Saccharomyces+cerevisiae+and+Schizosaccharomyces+pombe+lipidomics.">Toward one step analysis of cellular lipidomes using liquid chromatography coupled with mass spectrometry: application to Saccharomyces cerevisiae and Schizosaccharomyces pombe lipidomics.</a> Mol. Biosyst. 6, 1008-1017 (2010).</li><li>Farvid, M. S., Ng, T. W. K., Chan, D. C., Barrett, P. H. R., Watts, G. 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Hay una multitud de técnicas que se han desarrollado para medir los niveles de lípidos 8-10. Sin embargo, cada uno de estos métodos viene con algunas desventajas importantes que ya están contemplados en el ensayo basado en la flotabilidad se describe anteriormente. En primer lugar, este ensayo es extremadamente rápido. Prueba de un gradiente de concentración completa tarda no más de 30 - 60 min. Esta es una gran mejora en la mayoría de las técnicas actualmente en uso. Por ejempl...

Drosophila melanogaster se ha utilizado durante más de un siglo en el estudio de la genética y otras cuestiones biológicas básicas. En las últimas décadas, se ha hecho evidente que la Drosophila es una poderosa herramienta en el estudio de muchas enfermedades humanas. Un 70 - 80% de los genes asociados con enfermedades humanas tienen un ortholog identificado mosca de 1 - 4, Drosophila proporciona un sistema simplificado aún traducible en el que estudiar las enfermedades complejas. Metabolismo, en particular, se ha beneficiado de estos estudios. No sólo son la genética del metabolismo bien conservados entre las moscas y los seres humanos, pero los órganos y tipos de células son también muy similares 2,5. Por ejemplo, la grasa corporal de las tiendas de la mosca de ambos triacilglicéridos (TAG) y glucógeno, funciones análogas a las realizadas en el hígado de los mamíferos y el tejido adiposo blanco 6. Uso de Drosophila como modelo para la obesidad humana ha mejorado enormemente nuestro conocimiento de los lípidosmetabolismo y la genética de la obesidad 7. Las larvas de desarrollo es particularmente útil para estudiar la segregación de los nutrientes en el almacenamiento o utilización, ya que está dedicado a la alimentación y el almacenamiento de energía que se utilizará durante pupariation. Actualmente, hay muchos diferentes métodos cuantitativos de determinación de los niveles de almacenamiento de grasa en Drosophila. El método más ampliamente utilizado es el ensayo colorimétrico acoplado (CCA) 8,9. CCA fue desarrollado para determinar los niveles de TAG en el suero humano y opera en la premisa de que el glicerol liberado de la columna vertebral de los triglicéridos se someterá a varias reacciones, lo que se traduce en una reacción redox acoplado a la generación de un producto coloreado. A continuación se mide la absorbancia de las longitudes de onda específicas de luz para determinar la cantidad inicial de glicerol presente. Sin embargo, el glicerol también puede ser liberada de mono y diacilglicéridos Además de TAG y por lo tanto puede no ser una medida precisa de sgrasa corporal reada 9. Por otra parte, pigmento de los ojos de las moscas adultas trituradas puede interferir con algunas lecturas de absorbancia y complicar los resultados 9,10. Por lo tanto, CCA debe ir acompañada y validado por cromatografía en capa fina (TLC), que permite la separación de la mayoría de las familias de lípidos que se puede cuantificar mediante densitometría 10,11. Sin embargo, algunas clases de lípidos como esteroles no pueden ser analizados y deben cuantificarse de manera diferente 12. Espectrometría de masas (MS) es una forma precisa para cuantificar todas las clases de los principales lípidos celulares 12,13. Sin embargo, los procedimientos de extracción de lípidos necesarias para analizar por MS consumen mucho tiempo (más la toma de casi un día completo) y costoso. Aquí presentamos un método alternativo para determinar rápida y barata niveles de grasa del organismo en las larvas L3 de Drosophila melanogaster. El método presentado a continuación explota la diferencia de densidad entre el tejido de grasa y tejido magro. fa mamíferost tejido tiene una densidad de aproximadamente 0,9 g / ml 14 mientras que el músculo esquelético como una densidad de 1,06 g / ml 15. Esta diferencia significa que los animales con las tiendas más altos de grasa tienen menor densidad que los animales con las tiendas más bajos de grasa, que les permitan una mejor flotan en una solución de densidad fija. Esta propiedad permite la detección extremadamente rápida de un gran número de animales, mientras que ser barato y no invasivo. Análisis basado en la flotabilidad se ha utilizado tanto para confirmar los fenotipos de la alteración de los reguladores conocidos de los niveles de grasa, así como para identificar nuevos reguladores genéticos y neurológicos de la obesidad 16,17.

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			<td height="21" style="height:21px;width:216px;">Bacto Agar</td>
			<td style="width:168px;">VWR</td>
			<td style="width:119px;">214030</td>
			<td style="width:167px;"></td>
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			<td height="42" style="height:42px;width:216px;">Welch&#39;s Original 100% Grape Juice</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sucrose</td>
			<td style="width:168px;">Fisher</td>
			<td style="width:119px;">S512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tegosept</td>
			<td style="width:168px;">Genesee Scientific</td>
			<td style="width:119px;">20-259</td>
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			<td height="21" style="height:21px;width:216px;">Ethanol</td>
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			<td>200 proof</td>
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			<td height="21" style="height:21px;width:216px;">Baker&#39;s yeast</td>
			<td style="width:168px;">Fleichmann&#39;s</td>
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			<td height="21" style="height:21px;width:216px;">Yellow Corn Meal</td>
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			<td>Enriched degerminated</td>
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			<td height="21" style="height:21px;width:216px;">Drosophila Agar Type II</td>
			<td style="width:168px;">Apex</td>
			<td style="width:119px;">66-104</td>
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			<td height="42" style="height:42px;width:216px;">Soy Flour</td>
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			<td height="21" style="height:21px;width:216px;">Malt Extract</td>
			<td style="width:168px;">Breiss</td>
			<td style="width:119px;">5748</td>
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			<td height="21" style="height:21px;width:216px;">Light Corn Syrup</td>
			<td style="width:168px;">Karo</td>
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			<td height="21" style="height:21px;width:216px;">Propionic Acid</td>
			<td>VWR</td>
			<td>U330-09</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
			<td style="width:168px;">Fisher</td>
			<td style="width:119px;">50147491</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Potassium phosphate monobasic</td>
			<td>Sigma</td>
			<td>P5655-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium phosphate anhydrous</td>
			<td style="width:168px;">Fisher</td>
			<td style="width:119px;">S3933</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium chloride</td>
			<td>Fisher</td>
			<td>P330-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 x 10mm petri plate</td>
			<td style="width:168px;">Fisher</td>
			<td>08-757-100A</td>
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			<td height="21" style="height:21px;width:216px;">50mL Conical</td>
			<td style="width:168px;">Fisher</td>
			<td style="width:119px;">50-869-569</td>
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</table>

a buoyancy-based method of determining fat levels in drosophila

Drosophila melanogaster is a key experimental system in the study of fat regulation. Numerous techniques currently exist to measure levels of stored fat in Drosophila, but most are expensive and/or laborious and have clear limitations. Here, we present a method to quickly and cheaply determine organismal fat levels in L3 Drosophila larvae. The technique relies on the differences in density between fat and lean tissues and allows for rapid detection of fat and lean phenotypes. We have verified the accuracy of this method by comparison to body fat percentage as determined by neutral lipid extraction and gas chromatography coupled with mass spectrometry (GCMS). We furthermore outline detailed protocols for the collection and synchronization of larvae as well as relevant experimental recipes. The technique presented below overcomes the major shortcomings in the most widely used lipid quantitation methods and provides a powerful way to quickly and sensitively screen L3 larvae for fat regulation phenotypes while maintaining the integrity of the larvae. This assay has wide applications for the study of metabolism and fat regulation using Drosophila.

La figura 1 representa un ejemplo de cómo el ensayo basado en la flotabilidad se puede detectar tanto las reservas de grasa más altos y más bajos en las larvas manipulado genéticamente. La Figura 1A muestra que la excitación o el silenciamiento de un cierto subconjunto de neuronas (E347) en el cerebro de las larvas induce grasa inferior y superior tiendas respectivamente. El mismo control antecedentes genéticos se utilizó en ambos casos. L...

Watch this Scientific Journal Video about A Buoyancy-based Method of Determining Fat Levels in Drosophila at JoVE.com

A Buoyancy-based Method of Determining Fat Levels in Drosophila

Aquí presentamos un método para medir los niveles de grasa organísmicos en el tercer estadio (L3) larvas de Drosophila melanogaster. Este método aprovecha la relativamente baja densidad de tejido graso para diferenciar entre las larvas con las reservas de grasa alterados. análisis basado en la flotabilidad es una herramienta valiosa para la detección rápida, reproducible y económico.

Un método basado en la flotabilidad de la determinación de los niveles de grasa en<em&gt; Drosophila</em

Drosophila melanogaster is a key experimental system in the study of fat regulation. Numerous techniques currently exist to measure levels of stored fat ...

The overall goal of this assay is to determine any changes in fat storage levels that may be caused by a change in genetics, diet, or treatment. These methods can help answer key questions in the field of metabolism such as genetic or neuronal contributions to obesity predisposition. The main advantage of this technique is that it's fast, economical, and very reproducible when compared to other methods currently used.Individuals new to this method or new to working with wandering stage larva may struggle with this protocol because the developmental timeline of different genotypes can be highly variable. To begin this procedure, add a small dollop of yeast paste to the middle of a grate plate. Smooth down any rough edges with a spatula.Next, transfer the flies of interest into an egg collection chamber, and place the grate plate with yeast paste on top facing inwards. Then tape the grate plate to the egg collection chamber to keep it from falling out. Make sure to write important genotype and date information on the bottom of the grate plate.Subsequently, upend the egg collection chamber to allow the flies to lay eggs on the grate plate. After that, cover the egg collection chamber with an open box and place the covered egg collection chamber in an incubator at 25 degrees celsius for four to six hours to allow the flies to lay eggs. When it is done, take the grate plate out of the egg collection chamber and transfer the flies back into their original bottle of food.Use a spatula to wipe off any excess yeast paste from the grate plate. Subsequently, store the grate plate in a larger Petri dish in a humidified incubator at 25 degrees celsius for 24 hours. In this procedure, score the top layer of the food by plunging it with a spatula several times to allow the larvae to burrow and feed.22 to 24 hours after egg collection use a small paintbrush to collect 50 first instar larvae of the same approximate size and place the larvae in the experimental food vial. It's very important that larvae of the same approximate size are selected and transferred to the experimental food. This will ensure tight coordination of larval development.Next, clean the paintbrush with a laboratory wipe and ensure that there are no remaining larvae on the paintbrush before continuing to another genotype. Afterward, place the vial of larvae in an incubator at 25 degrees celsius and allow them to develop. Remove the vial of larvae from the incubator once there are 20 to 40 larvae wandering up the sides of the vial.Record the date and time of the assay as well as the number of wandering larvae. Then prepare the experimental solution by adding 11.5 milliliters of PBS and nine milliliters of 20%sucrose to a 50 milliliter conical tube. Add 20%sucrose to the vial until it reaches one inch from the top of the vial.Use a spatula to gently stir the food top layer to free the larvae that are still burrowing in it. All the larvae will rise to the surface of the sucrose solution. Afterward, gently transfer the larvae into the initial sucrose solution and gently stir them.While transferring the larvae to the experimental solution, be careful not to squeeze or puncture the larvae. This may alter the results of the assay. Cap the conical tube and upend it several times to thoroughly mix the solution.Then uncap the conical tube and swirl it to create a gentle vortex. Allow two to five minutes for the larvae to settle, either floating to the top of the solution or sinking to the bottom. Record the number of floating larvae, and the specific sucrose concentration tested.Then add one milliliter of 20%sucrose to the experimental solution to increase its density. Subsequently, use a spatula to stir the larvae gently. Following this, cap the conical tube and upend it several times to thoroughly mix the solution.Subsequently, uncap the conical tube and swirl to create a gentle vortex. Record the number of floating larvae and the new concentration. Continue this process until at least 95%of the larvae are floating.Add 20%sucrose to the conical vial until it is one inch from the top. Next collect all the larvae in a dissection dish filled with PBS. Examine the larvae under a microscope for any small larvae, prepupae, pupae, or any other differences between genotypes to ensure that the procedures were done appropriately.Then record the total number of larvae. Afterward, transfer 10 larvae on a laboratory wipe for drying. Label a microcentrifuge tube and place the larvae in the tube.Subsequently, flash freeze the larvae in liquid nitrogen and store the tube at minus 20 degrees celsius. Shown here is an example of how the buoyancy-based assay can detect both higher and lower fat stored in the genetically manipulated larvae. This figure shows that excitation or silencing of a certain subset of neurons in the larval brain induces lower and higher stored fat respectively.The same genetic background control was used in both cases. Here is an example of how this phenotype obtained by the density assay is corroborated by neutral lipid extraction and quantification by GCMS. Once mastered, this technique can be done within 30 minutes if properly performed, and may be performed in tandem with other genotypes.While attempting this procedure, it's important to remember to synchronize the development of the experimental larvae as much as possible. Following this procedure, larvae can be snap frozen in liquid nitrogen to be used in other methods like gas chromatography mass spectrometry in order to obtain additional information like the specific classes of lipid types in the larvae. After its development, this technique paved the way for researchers in metabolism to explore the genetic and or neuronal contributions to obesity in Drosophila.After watching this video, you should have a good understanding of how to quickly screen genotypes for changes in levels of stored fats.

Research

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A Buoyancy-based Method of Determining Fat Levels in Drosophila

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Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Visually Mediated Odor Tracking During Flight in Drosophila

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 ...

In-vivo Centrifugation of Drosophila Embryos

In-vivo Centrifugation of Drosophila Embryos

En vivo-La centrifugación de embriones de Drosophila

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

Bioquímica y de Evaluación de Rendimiento microscópica de masa grasa en<em&gt; Caenorhabditis elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

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

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

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 ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Medición de niveles hemo síntesis en células de mamífero

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

Un método de permeabilización de<em&gt; Drosophila</em&gt; Los embriones para ensayos de moléculas pequeñas Actividad

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Cuantitativa inmunofluorescencia Ensayo para medir la variación en los niveles de proteína en centrosomas

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

La determinación de la proteína de membrana de topología usando fluorescencia de Protección de la proteasa (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...