Nos gustaría dar las gracias a la Sociedad Max-Planck para la financiación de nuestra investigación.

1. Preparación de respirómetros <ol><li> Corte la punta de la pipeta 1.000 l con una hoja de afeitar para permitir la inserción de los capilares de la micropipeta 50 l, tratar de conseguir la punta de la pipeta lo más recto posible. </li><li> Coloque un pedazo de espuma en la pipeta y presione hacia abajo en la punta de la pipeta. </li><li> Agregue una pequeña cantidad de absorbente de CO 2 y de contenerlo por una segunda pieza de espuma. </li><li> Aplicar el pegamento en el lugar donde se inserta la micropipeta en la punta de la pipeta. </li><li> Deje el respirómetro durante la noche para permitir que el pegamento se seque. Un diagrama esquemático de un respirómetro se muestra en la Figura 1A. </li></ol> 2. Preparación de la cámara de medición <ol><li> Preparar la solución de cámara de mezclando agua con eosina en una relación que se traducirá en la coloración visible. </li><li> Verter la solución de eosina / agua en la cámara. </li><li> Etiqueta de uno de los lados de la cámara con una escala de centímetros. </li></ol><ol><li> Etiquetar los respirómetros individuales con un marcador. </li><li> Anestesie moscas utilizando un método alternativo a CO 2 y colocar 3-5 moscas del genotipo deseado dentro de cada respirómetro. </li><li> Selle las respirómetros firmemente en la parte superior el uso de masilla de plastilina. </li><li> Permitir que las moscas se recuperen de la anestesia durante aproximadamente 15 min. </li><li> Preparar una respirómetro sin moscas, que se utilizó como control atmosférica. </li></ol> 4. Realización del experimento <ol><li> Cuelgue las respirómetros en la cámara uniendo un soporte de tubo Eppendorf de 1,5 ml que está abierto en la parte superior y la parte inferior en la parte superior de la cámara. </li><li> Inserte respirómetros con la punta de la micropipeta hacia la cámara que permite que la punta se sumerja en la solución de color. </li><li> Añadir vaselina entre la cubierta de la tapa y la cámara para proporcionar aislamiento más fuerte de temperatura y presión fluctluaciones. </li><li> Cierre la tapa y deje que el sistema se equilibre durante 15 min. </li><li> Tome una fotografía de la cámara de asegurarse de que el nivel de líquido dentro de cada micropipeta es visible y también lo es la escala (véase el ejemplo mostrado en la Figura 1B). </li><li> Después de 1-2 horas, tomar otra foto. </li><li> Cuando se termina el experimento, eliminar las moscas de respirómetros y sopesar si se desea o transferirlos de nuevo al frasco si es necesario más. </li></ol> 5. Análisis de los resultados <ol><li> Abrir las imágenes adquiridas utilizando el software ImageJ 11. </li><li> Utilizando la escala en cada imagen, ajuste la escala de píxeles en el software. </li><li> Medir la distancia (Dd) que el líquido viajó desde un punto de referencia determinado en las imágenes tomadas al principio (d1) y al final del experimento (d2). Un ejemplo de esquema se muestra en la figura 1C. </li><li> Calcular la cantidad de CO2 producido (l / h / mosca) con la fórmula: </li></ol><img alt="Figura 1" fo:content-width="2in" src="/files/ftp_upload/51681/51681eq1.jpg" /> R = radio del tubo micropipeta en centímetros Dd = distancia que el líquido se ha movido en la micropipeta de muestras de ensayo se mide en centímetros ? C = distancia que el líquido se ha movido en la micropipeta de la muestra de control negativo (sin moscas) n = número de moscas utilizadas h = horas

<ol>
	<li>Van Voorhies, W. A., Khazaeli, A. A., Curtsinger, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+the+&amp;#34;rate+of+living&amp;#34;+model:+further+evidence+that+longevity+and+metabolic+rate+are+not+inversely+correlated+in+Drosophila+melanogaster.">Testing the &amp;#34;rate of living&amp;#34; model: further evidence that longevity and metabolic rate are not inversely correlated in Drosophila melanogaster.</a> J Appl Physiol. 97, 1915-1922 (2004).</li><li>Ross, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-specific+decrease+in+aerobic+efficiency+associated+with+increase+in+oxygen+free+radical+production+in+Drosophila+melanogaster.">Age-specific decrease in aerobic efficiency associated with increase in oxygen free radical production in Drosophila melanogaster.</a> Journal of Insect Physiology. 46, 1477-1480 (2000).</li><li>van Voorhies, W. A., Khazaeli, A. A., Curtsinger, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selected+contribution:+long-lived+Drosophila+melanogaster.+lines+exhibit+normal+metabolic+rates.">Selected contribution: long-lived Drosophila melanogaster. lines exhibit normal metabolic rates.</a> J Appl Physiol. 95, 2605-2613 (2003).</li><li>Hulbert, A. J., 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=Metabolic+rate+is+not+reduced+by+dietary-restriction+or+by+lowered+insulin/IGF-1+signalling+and+is+not+correlated+with+individual+lifespan+in+Drosophila+melanogaster.">Metabolic rate is not reduced by dietary-restriction or by lowered insulin/IGF-1 signalling and is not correlated with individual lifespan in Drosophila melanogaster.</a> Experimental Gerontology. 39, 1137-1143 (2004).</li><li>Ueno, T., Tomita, J., Kume, S., Kume, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+modulates+metabolic+rate+and+temperature+sensitivity+in+Drosophila+melanogaster.">Dopamine modulates metabolic rate and temperature sensitivity in Drosophila melanogaster.</a> PLoS ONE. 7, (2012).</li><li>Takeuchi, K., 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=Changes+in+temperature+preferences+and+energy+homeostasis+in+dystroglycan+mutants.">Changes in temperature preferences and energy homeostasis in dystroglycan mutants.</a> Science. 323, 1740-1743 (2009).</li><li>Kucherenko, M. M., Marrone, A. K., Rishko, V. M., Magliarelli Hde, F., Shcherbata, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+and+muscular+dystrophy:+a+genetic+screen+for+dystroglycan+and+dystrophin+interactors+in+Drosophila.+identifies+cellular+stress+response+components.">Stress and muscular dystrophy: a genetic screen for dystroglycan and dystrophin interactors in Drosophila. identifies cellular stress response components.</a> Developmental Biology. 352, 228-242 (2011).</li><li>Marrone, A. K., Kucherenko, M. M., Wiek, R., Gopfert, M. C., Shcherbata, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperthermic+seizures+and+aberrant+cellular+homeostasis+in+Drosophila+dystrophic.+muscles.">Hyperthermic seizures and aberrant cellular homeostasis in Drosophila dystrophic. muscles.</a> Scientific Reports. 1, 47 (2011).</li><li>Khazaeli, A. A., Van Voorhies, W., Curtsinger, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longevity+and+metabolism+in+Drosophila+melanogaster:+genetic+correlations+between+life+span+and+age-specific+metabolic+rate+in+populations+artificially+selected+for+long+life.">Longevity and metabolism in Drosophila melanogaster: genetic correlations between life span and age-specific metabolic rate in populations artificially selected for long life.</a> Genetics. 169, 231-242 (2005).</li><li>Elia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+equivalents+of+CO2+and+their+importance+in+assessing+energy+expenditure+when+using+tracer+techniques.">Energy equivalents of CO2 and their importance in assessing energy expenditure when using tracer techniques.</a> The American Journal of Physiology. 260, (1991).</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9, 676-682 (2012).</li><li>Bharucha, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epicurean+fly:+using+Drosophila+melanogaster.+to+study+metabolism.">The epicurean fly: using Drosophila melanogaster. to study metabolism.</a> Pediatric Research. 65, 132-137 (2009).</li><li>Rajan, A., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+flies+and+men:+insights+on+organismal+metabolism+from+fruit+flies.">Of flies and men: insights on organismal metabolism from fruit flies.</a> BMC Biology. 11, 38 (2013).</li></ol>

No tenemos nada que revelar.

En este protocolo, se describe un método de bajo costo y confiable para medir la producción de CO 2 en las moscas. Hemos encontrado que este experimento es fácil, rápido de realizar y genera datos reproducible que está de acuerdo con otros estudios 1, 6, 9. El protocolo descrito aquí se puede modificar fácilmente para adaptarse a presupuesto y materiales disponibles de cualquier laboratorio. La construcción de cada respirómetro individuo puede adaptarse, siempre y cuando la cámara permane...

El ciclo bioquímico de Krebs genera ATP a través de la oxidación de acetato derivado de los carbohidratos, las grasas y las proteínas que producen CO 2. En Drosophila, O 2 de entrada está directamente relacionada con la producción de CO 2 y refleja el nivel de metabolismo 1. Por lo tanto, la medición de CO 2 de salida ha utilizado con éxito en los estudios relacionados con el envejecimiento y el metabolismo de 2-5. Aquí nuestro laboratorio ha modificado montajes experimentales previamente diseñados, lo que permite la medición de la producción de CO 2 en un máximo de dieciocho muestras sin necesidad de equipo especializado. Otros y hemos utilizado anteriormente este método para mostrar las diferencias en las tasas metabólicas en las moscas que son deficientes en la proteína de la distrofia muscular asociada, Distroglicano (Dg) 6-8. O 2 utilizado para el metabolismo oxidativo se convierte en CO 2, que es expulsado como residuos respiratoria. La construcciónción de respirómetros hechas a mano se describe que permite la determinación de la tasa de O 2 consumido. Las moscas se colocan en un recipiente sellado con una sustancia que absorbe CO 2 expulsado, eliminando de manera eficiente desde la fase gaseosa. El cambio en el volumen de gas (disminución de la presión) se mide por el desplazamiento de fluido en un capilar de vidrio unido a la respirómetro cerrado. La principal ventaja de esta técnica sobre otros es el costo. Estudios previos han medido la producción de CO2 por Drosophila utilizando analizadores de gases y sistemas de respirometría técnicamente avanzados 1,9. A pesar de el equipo más complejo, la sensibilidad del método descrito aquí es similar a los valores reportados (Tabla 1). Además, varios otros grupos han utilizado variaciones de esta técnica para determinar las tasas metabólicas relativas en Drosophila 4-6. Por lo tanto, este ensayo se puede usar para generar reliable, datos reproducibles relevantes para el metabolismo de Drosophila sin la compra de equipo especializado que puede ser configurado en cualquier laboratorio y se puede utilizar con fines educativos. En general, las técnicas aceptadas para determinar el metabolismo de un organismo es medir el CO 2 producido, el O 2 consumido, o ambos 3,4,9. Aunque, se puede suponer que un equivalente de O 2 genera un equivalente de CO 2, la relación precisa de CO 2 generado es dependiente sobre el sustrato metabólico utilizado 10. Por lo tanto, para determinar con precisión la tasa metabólica en unidades de energía que es necesario medir ambos O 2 consumido y CO 2 producido. Debido a esto, el método descrito aquí es específicamente relevante para la comparación de las diferencias en la producción de CO 2 entre los animales y no el valor absoluto. Nuestra técnica se integra animales CO producción múltiple 2 durante un período de timE (1-2 horas) y por lo tanto devuelve un promedio de la actividad de los animales. Si hay razones para creer que los animales de experimentación son menos activos que los animales de control de la medición podría reflejar diferentes niveles de actividad y no necesariamente el metabolismo.

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			<td height="21" style="height:21px;width:401px;">BlauBrand IntraMark 50&#181;l micropipettes</td>
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			<td style="width:119px;">612-1413</td>
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			<td height="21" style="height:21px;width:401px;">Soda Lime</td>
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			<td height="21" style="height:21px;width:401px;">Eosine&#160;</td>
			<td style="width:101px;">Sigma&#160;</td>
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			<td>Any dye that can create visible colorization of liquid can be used</td>
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			<td height="22" style="height:22px;width:401px;">Thin Layer Chromatorgaphy (TLC) Developing Chamber</td>
			<td style="width:101px;">VWR</td>
			<td>21432-761</td>
			<td>Any transparent glass chamber that can be closed with the lid</td>
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			<td height="21" style="height:21px;">Anesthetizer, Lull-A-Fly Kit</td>
			<td style="width:101px;">Flinn</td>
			<td>FB1438</td>
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			<td height="21" style="height:21px;width:401px;">1 ml pipett tips</td>
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			<td style="width:101px;">Any</td>
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			<td height="21" style="height:21px;">Plaesticine Putty</td>
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			<td height="21" style="height:21px;width:401px;">Scalpel</td>
			<td style="width:101px;">Any</td>
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			<td height="21" style="height:21px;width:401px;">Twezzers&#160;</td>
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measurement of metabolic rate in drosophila using respirometry

Trastornos metabólicos son un problema frecuente que afecta a la salud humana. Por lo tanto, la comprensión de los mecanismos que regulan el metabolismo es una tarea científica fundamental. Muchos de los genes que causan enfermedades en los seres humanos tienen un homólogo mosca, haciendo Drosophila un buen modelo para estudiar las vías de señalización implicadas en el desarrollo de diferentes trastornos. Además, la maleabilidad de Drosophila simplifica pantallas genéticos para ayudar en la identificación de nuevas dianas terapéuticas que puedan regular el metabolismo. Con el fin de realizar una pantalla de este tipo es necesario un método sencillo y rápido para identificar los cambios en el estado metabólico de las moscas. En general, la producción de dióxido de carbono es un buen indicador de los gastos de oxidación del sustrato y la energía que proporciona información sobre el estado metabólico. En este protocolo se introduce un método simple para medir el CO 2 de salida de las moscas. Esta técnica potencialmente puede ayudar en la identificación de las perturbaciones genéticos que afectan la tasa metabólica.

Con el fin de demostrar que el método es sensible que mide la producción de CO 2 de tipo salvaje (Oregon R) macho vuela a 18, 25 y 29 ° C y las moscas mutantes para Dg. Las moscas se elevaron a 25 ° C y luego se desplazan a la temperatura experimental durante 5 días antes de la medición. Como era de esperar para esta especie ectotérmico, la cantidad de CO 2 producido aumentó con la temperatura (Figura 2). Hemos en el pasado muestran que una dieta libre de ...

Watch this Scientific Journal Video about Measurement of Metabolic Rate in Drosophila using Respirometry at JoVE.com

Measurement of Metabolic Rate in Drosophila using Respirometry

Los trastornos metabólicos se encuentran entre una de las enfermedades más comunes en los seres humanos. El modelo de organismo genéticamente manejable D. melanogaster se puede utilizar para identificar nuevos genes que regulan el metabolismo. Este artículo describe un método relativamente sencillo que permite el estudio de la tasa metabólica en las moscas de la medición de su producción de CO 2.

La medición de la tasa metabólica en<em&gt; Drosophila</em&gt; Mediante respirometría

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

measurement-of-metabolic-rate-in-drosophila-using-respirometry

Research

JoVE Journal

Biology

Measurement of Metabolic Rate in Drosophila using Respirometry

19.4K vistas.  Max Planck Institute for Biophysical Chemistry.  Los trastornos metabólicos se encuentran entre una de las enfermedades más comunes en los seres humanos. El modelo de organismo genéticamente manejable D. melanogaster se puede utilizar para identificar nuevos genes que regulan el metabolismo. Este artículo describe un método relativamente sencillo que permite el estudio de la tasa metabólica en las moscas de la medición de su producción de CO 2. 

Video: Measurement of Metabolic Rate in Drosophila using Respirometry

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

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Medición de la esperanza de vida en<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

Un método de alto rendimiento para la medición de la tasa de filtración glomerular en ratones conscientes

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

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 ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

La medición de las larvas La actividad en el<em&gt; Drosophila</em&gt; Monitor de Actividad

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Medición y Análisis de la producción de ácido extracelular para Determinar glicolítica Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Measurement of Carbon Dioxide Production from Radiolabeled Substrates in Drosophila melanogaster

The power of Drosophila genetics is increasingly being applied to questions of hormone signaling and metabolism and to the development of models of human disease in this organism. Sensitive methods for measurements of parameters such as metabolic rates are needed to drive the understanding of physiology and disease in small animals such as the fruit fly. The method described here assesses fuel oxidation in small numbers of adult flies fed food containing trace amounts of 14C-labeled substrates such as glucose or fatty acid. After the feeding period and any additional experimental manipulations, flies are transferred to short tubes capped with mesh, which are then placed in glass vials containing KOH-saturated filter paper that traps exhaled, radiolabeled CO2 generated from oxidation of radiolabeled substrates as potassium bicarbonate, KHCO3. This radiolabeled bicarbonate is measured by scintillation counting. This is a quantitative, reproducible, and simple approach for the study of fuel oxidation. The use of radiolabeled glucose, fatty acids, or amino acids allows determination of the contribution of these different fuel sources to energy metabolism under different conditions such as feeding and fasting and in different genetic backgrounds. This complements other approaches used to measure in vivo energy metabolism and should further the understanding of metabolic regulation.

Measurement of Carbon Dioxide Production from Radiolabeled Substrates in Drosophila melanogaster

This paper describes a method for the measurement of fuel oxidation in Drosophila melanogaster in which trace amounts of specific radiolabeled metabolic substrates are fed to flies. The exhaled radiolabeled CO2 that is a produced from fuel oxidation is collected and measured.

La medición de la producción de dióxido de carbono a partir de sustratos radiomarcados en<em&gt; Drosophila melanogaster</em

The power of Drosophila genetics is increasingly being applied to questions of hormone signaling and metabolism and to the development of models of human ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

Mezcla de cultivos primarios de murino intestino delgado previstos para el Estudio de Gut Hormone secreción y Imágenes de células vivas de células enteroendocrinas

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

Medición del metabolismo energético en el tejido retiniano Explanted utilizando el análisis de flujo extracelular

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

High-Resolution Respirometry to Assess Bioenergetics in Cells and Tissues Using Chamber- and Plate-Based Respirometers

High-resolution respirometry (HRR) allows monitoring oxidative phosphorylation in real-time for analysis of individual cellular energy states and assessment of respiratory complexes using diversified substrate-uncoupler-inhibitor titration (SUIT) protocols. Here, the usage of two high-resolution respirometry devices is demonstrated, and a basic collection of protocols applicable for the analysis of cultured cells, skeletal and heart muscle fibers, and soft tissues such as the brain and liver are presented. Protocols for cultured cells and tissues are provided for a chamber-based respirometer and cultured cells for a microplate-based respirometer, both encompassing standard respiration protocols. For comparative purposes, CRISPR-engineered HEK293 cells deficient in mitochondrial translation causing multiple respiratory system deficiency are used with both devices to demonstrate cellular defects in respiration. Both respirometers allow for comprehensive measurement of cellular respiration with their respective technical merits and suitability dependent on the research question and model under study.

Assessing oxidative phosphorylation using high-resolution respirometers has become an integral part of the functional analysis of mitochondria and cellular energy metabolism. Here, we present protocols for the analysis of cellular energy metabolism using chamber and microplate-based high-resolution respirometers and discuss the key benefits of each device.