Name: high throughput danio rerio energy expenditure assay
Uploaded: 2016-01-27T17:00:00.000+00:00
Duration: 8 min 35 s
Description: Watch this Scientific Journal Video about High Throughput Danio Rerio Energy Expenditure Assay at JoVE.com

The authors would like to thank Drs. Roger Cone and Chao Zhang for their contributions to validating the application of this assay as previously published. This work was supported by NIH 1F32DK082167-01 (BJR).

Note: This protocol follows the guidelines of the University of Arizona Office for the Responsible Conduct of Research and has been approved by the Institutional Animal Care and Use Committee
1. Zebrafish Breeding and Embryo Maintenance
<ol>
	<li>Prepare E3 embryo medium8. To Make 60x stock solution, dissolve 34.8 g NaCl, 1.6 g&#160;KCl, 5.8g CaCl2&#9679;2H2O, and 9.78 g MgCl2&#9679;6H2O in 1.95 L ddiH2O. Adjust pH to 7.2 using NaOH and HCl Adjust volume to 2 L using ddiH2O.</li>
	<li>To prepare 1x E3 embryo medium, dilute 16.5 ml 60x stock in 1 L ddiH20. Add 100 &#181;l 1% methylene blue.</li>
	<li>Breeding
	<ol>
		<li>House Male and Female brood stock (7 - 18 months of age) separately to maximize fecundity.</li>
		<li>Turn the lights off 1 - 2 hr before on the day before eggs are desired. Set up mating (1 male and 1 - 2 females) in crossing cages as previously described9. Crossing cages allow for the eggs to escape predation as they pass through the perforated base of the tank insert in which the adult fish are held.</li>
		<li>Optional: For timed breeding, (as would be needed for studies involving morpholino injection), separate the male and female fish by a clear plastic divider., Remove this in the morning to result in timed laying of the eggs and fertilization. For morpholino studies, perform power calculations to assess the number of embryonic fish required per treatment. Without an estimate of the magnitude of effect or variation, a preliminary trial can be run with an n = 8.</li>
	</ol>
	</li>
	<li>Pour the eggs from the crossing cage into a petri dish (100 - 150 embryos/10 cm petri dish). Allow the eggs to settle to the bottom of the dish and pour off the water. Remove any remaining water using a fine tip disposable transfer pipette. Add back E3 embryo medium made in step 1.1.</li>
	<li>Make a flame polished disposable pipette for future egg and fish transfer. Using scissors, cut a graduated disposable transfer pipette at the 0.25 ml graduation. To remove rough edges, flame polish the cut tip over a Bunsen burner.</li>
	<li>Maintain embryonic fish to 4 days post-fertilization (dpf) in 10 cm petri dishes in zebrafish E3 embryo medium at 28.5 &#9675;C. Twice each day, use a flame polished graduated disposable transfer pipette to remove any unfertilized eggs or dead embryos (opaque white appearance). Once each day, replace E3 embryo medium. Pour off E3 embryo medium, remove remaining liquid using a fine tip disposable transfer pipette, and add back pre-warmed (28.5 &#9675;C) E3 embryo medium.</li>
</ol>
2. Prepare Assay Solution
<ol>
	<li>Prepare the assay solution according to Table 1. Sterile filter assay solution using a 0.22 &#181;M filter. Prior to assay initiation, warm the sterile assay solution in a 28.5 &#9675;C water bath.</li>
</ol>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Ingredient</td>
			<td>% of Total</td>
			<td>Volume (&#181;l/10 ml)</td>
		</tr>
		<tr>
			<td>60x E3 Embryo Medium</td>
			<td>1.667</td>
			<td>166.7</td>
		</tr>
		<tr>
			<td>Double deionized (ddi) H20</td>
			<td>96.233</td>
			<td>9623.3</td>
		</tr>
		<tr>
			<td>Alamar Blue</td>
			<td>1</td>
			<td>100</td>
		</tr>
		<tr>
			<td>400 mM NaOH</td>
			<td>1</td>
			<td>100</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>0.1</td>
			<td>10</td>
		</tr>
	</tbody>
</table>
Table 1. Assay Medium
3. Perform Assay
<ol>
	<li>Rinse zebrafish embryos 3 times with sterile filtered E3 embryo medium. Combine all viable zebrafish embryos from the clutch into a beaker. Sterile filter 75 ml E3 embryo medium. Using a fine tip disposable transfer pipette, remove as much of the E3 embryo medium from the zebrafish embryos as possible. Add back 20 ml sterile filtered E3 embryo medium. Remove and replace E3 embryo medium 3 times.</li>
	<li>Plate 1 fish into each of 93 wells of a 96-well black sided clear bottom plate. Transfer the embryonic fish to the plate by individually capturing an embryo within a flame polished graduated plastic disposable transfer pipette (step 1.5) and gently ejecting into the well. Transfer E3 embryo medium taken from the beaker containing rinsed fish into the 3 blank wells. This ensures that blank wells have been exposed to the exact same water as the wells that contain fish. Both wells that contain fish and blank wells should be at least &#189; full (200 &#181;l) at the conclusion of this step.</li>
	<li>Replace E3 embryo medium with assay solution in all 96 wells, including blank wells. This step should be performed 1 column at a time for each of the 12 columns in a 96 well plate. Using a fine tip disposable transfer pipette, remove E3 embryo medium from every well in one column of the plate (8 wells). Take care to not touch the zebrafish embryo. Add 300 &#181;l&#160;of assay solution to each well from which water was removed. Repeat the removal of E3 embryo medium and replacement with assay solution for all 12 columns of the plate.</li>
	<li>Optional: To test the metabolic rate change in response to a compound, make a solution that is 100 times (100X) the desired treatment concentration. Of note, ensure that the 100x solution does not exceed 10% DMSO to limit the final DMSO concentration to no more than 0.1%. Add 3 &#181;l of the 100x solution to each of the treatment wells. 
	NOTE: Perform power calculations to assess the number of treatment wells. However, without an estimate of the magnitude of response or variation, a preliminary trial can be run in 8 compound treated and 8 vehicle treated control wells that contain fish.
	<ol>
		<li>Add 3 &#181;l of vehicle control to control (no drug treatment) wells that contain fish. To ensure that the fluorescent response to compound is a result of actions within the fish, apply drug and vehicle treatments to 3 blank wells in a 96 well plate.</li>
	</ol>
	</li>
	<li>Read fish filled plate on fluorescent plate reader with excitation wavelength at 530 nM and emission wavelength at 590 nM.</li>
	<li>Place plate into humidified incubator set to 28.5 &#9675;C (maintain plate in darkness throughout assay to avoid photobleaching of assay solution). The assay can be run for durations of time that range from 1 hr to 24 hr. The duration of assay depends on the objective of the study. For testing the response to a short acting non-stable compound a short duration may be most suitable. Yet, for stable compound tests or tests of genetic effects, maximizing assay duration is preferable to exploit the advantages associated with the cumulative signal.</li>
	<li>At a pre-determined time point (see step 3.6 to help determine timing) read fluorescence of fish-filled plate again with the same settings as step 3.5.</li>
</ol>
4. Assess the Relative Change in Fluorescence Associated with Treatment
<ol>
	<li>Calculate change in fluorescence of each well by subtracting the fluorescence at time 0 from the fluorescence at the conclusion of the study. Wells without fish (blanks) should have values near 0, far below values from wells that contain fish. 
	&#160;&#160;&#160;&#160;&#160;&#160;&#160; Change in Fluorescence = Fluorescence at conclusion - Fluorescence at time 0</li>
	<li>To ascertain the relative change in fluorescence, calculate the average change in fluorescence for control wells then divide the change in fluorescence of each well by the average change in fluorescence of the control wells. Of note, controls could be either vehicle controls or genetic controls. 
	&#160;&#160;&#160;&#160;&#160;&#160;&#160; <img alt="Equation 1" src="/files/ftp_upload/53297/53297eq1.jpg" /></li>
</ol>

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The authors have no competing financial interests to disclose.

Contamination with bacteria or fungi will greatly limit application of this assay. The methylene blue in the E3 embryo medium limits the possibility of fungal contamination. Throughout embryo rearing it is critical that care is taken to remove dead embryos twice each day. Additionally, it is essential to thoroughly rinse the embryos with sterile E3 embryo medium on the day of assay initiation. These 2 steps limit the potential for bacterial contamination of the embryos. The inclusion of blank wells described in step 3.2 ...

The mouse is currently the predominant model for obesity research. The short generation interval and genetic tools available in the mouse have been unmatched to date. However, the zebrafish also has a short generation interval (3 - 4 months) and surpasses even the mouse in ease of genetic manipulation 1,2. The zebrafish maintains nearly 90% of mammalian genes, while far exceeding the mouse in number of offspring and potential for use in genetic and drug screens 3.
To tap the potential of the zebrafish model for studies in obesity, assays must be developed to investigate factors that influence body weight regulation, including energy expenditure. As metabolites are processed through &#946;-oxidation and the tricarboxylic acid cycle oxygen is consumed and NADH2 is produced. Thus, NADH2 is a direct indicator of the flux of metabolites through metabolic pathways (metabolite oxidation). In poikilotherms, H+ leak through the inner mitochondrial membrane, which uncouples NADH2 oxidation from ATP synthesis, is 4 - 5 fold lower than in homeotherms 4. Accordingly, in zebrafish NADH2 is very tightly linked to ATP production through oxidative phosphorylation. Herein, we describe an assay that measures NADH2 production in larval zebrafish as a proxy for energy expenditure5.
Oxygen consumption is the gold standard for measuring energy expenditure. Yet, to best take advantage of the high throughput potential of the zebrafish, assays of energy expenditure must be amenable to high-throughput. Oxygen consumption systems that depend on a closed chamber circulating system are limited in throughput by the number of chambers available6. Open air O2 consumption/CO2 production assays have also been applied in the zebrafish5,7. These open air 96-well plate based systems are amenable to high throughput. Unfortunately, gas exchange with the environment limits sensitivity of these assays. We recently published the application of an assay that monitors NADH2 using the redox indicator alamarBlue5. This assay overcomes the limitations in throughput and sensitivity common to analyses of oxygen consumption in the zebrafish.
The zebrafish is becoming an increasingly important model for studies of whole body energy homeostasis. In part, because zebrafish are amenable to use in forward genetic screens and drug screens. Moreover, targeted genetic manipulation, including knockdown and knock-in, can be quickly applied. We have previously shown that this assay can be combined with bath drug application and genetic knockdown or knockout to identify compounds and genes that alter metabolic rate5. Moreover, this assay is designed to exploit the high throughput advantages inherent to the zebrafish.

<table><tbody><tr><td>AlamarBlue</td><td>Fisher Scientific</td><td>10-230-103</td><td>Manufactured by Thermo Scientific</td></tr><tr><td>Fine Tip DisposableTransfer Pipette</td><td>Fisher Scientific</td><td>13-711-26</td><td>Manufactured by Thermo Scientific</td></tr><tr><td>Fisherbrand Graduated Disposable Transfer Pipette</td><td>Fisher Scientific</td><td>13-771-9AM</td><td>Manufactured by Thermo Scientific</td></tr><tr><td>Black sided clear bottom 96-well plates</td><td>Fisher Scientific</td><td>50-320-785</td><td>Manufactured by E&amp;amp;K Scientific Products Inc.</td></tr></tbody></table>

high throughput danio rerio energy expenditure assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

Assay solution does not increase fluorescence in the absence of embryonic fish (Figure 1). However, the assay is highly sensitive to small changes in metabolic rate within the well. A single fish within a well creates a signal significantly different from blank wells within 1 hr (P&#60;0.0001). Figure 2 provides a visual representation of signals generated by embryonic fish after 24 hr of incubation. For the purpose of this picture clear sided wells were used. Yet, black sided wells are ...

Watch this Scientific Journal Video about High Throughput Danio Rerio Energy Expenditure Assay at JoVE.com

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

high-throughput-danio-rerio-energy-expenditure-assay

Research

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Biology

12.6K Views.  Vanderbilt University Medical Center. Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery. 

Video: High Throughput Danio Rerio Energy Expenditure Assay

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

Genetics

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, Drosophila research lacks popularity in developing countries, possibly due to the misinformed idea that establishing a lab and performing relevant experiments with such tiny insects is difficult and requires expensive, specialized apparatuses. Here, we describe how to build an affordable flylab to quantitatively analyze a myriad of behavioral parameters in D. melanogaster, by 3D-printing many of the necessary pieces of equipment. We provide protocols to build in-house vial racks, courtship arenas, apparatuses for locomotor assays, etc., to be used for general fly maintenance and to perform behavioral experiments using adult flies and larvae. We also provide protocols on how to use more sophisticated systems, such as a high resolution oxygraph, to measure mitochondrial oxygen consumption in larval samples, and show its association with behavioral changes in the larvae upon the xenotopic expression of the mitochondrial alternative oxidase (AOX). AOX increases larval activity and mitochondrial leak respiration, and accelerates development at low temperatures, which is consistent with a thermogenic role for the enzyme. We hope these protocols will inspire researchers, especially from developing countries, to use Drosophila to easily combine behavior and mitochondrial metabolism data, which may lead to information on genes and/or environmental conditions that may also regulate human physiology and disease states.

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

We provide protocols for anyone with a "maker culture" mind to start building a flylab for quantitative analysis of a myriad of behavioral parameters in Drosophila melanogaster, by 3D-printing many of the necessary pieces of equipment. We also describe a high resolution respirometry protocol using larvae to combine behavioral and mitochondrial metabolism data.

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, ...

Behavior

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental factors, and the toxicological and pharmacological effects of numerous substances. Few methods currently implemented are amenable to high throughput measurement. The Microplate Feeder Assay (MFA) was developed for quantifying the consumption of liquid food for individual flies using absorbance. In this assay, flies consume liquid food medium from select wells of a 1536-well microplate. By incorporating a dilute tracer dye into the liquid food medium and loading a known volume into each well, absorbance measurements of the well acquired before and after consumption reflect the resulting change in volume (i.e., volume consumed). To enable high throughput analysis with this method, a 3D-printed coupler was designed that allows flies to be sorted individually into 96-well microplates. This device precisely orients 96- and 1536-well microplates to give each fly access to up to 4 wells for consumption, thus enabling food preference quantification in addition to regular consumption. Furthermore, the device has barrier strips that toggle between open and closed positions to allow for controlled containment and release of a column of samples at a time. This method enables high throughput measurements of consumption of aqueous solutions by many flies simultaneously. It also has the potential to be adapted to other insects and to screen consumption of nutrients, toxins, or pharmaceuticals.

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

The microplate feeder assay offers an economical, high throughput method for quantifying liquid food consumption in Drosophila. A 3D-printed device connects a 96-well microplate in which flies are housed to a 1536-well microplate from which flies consume a feeding solution with a tracer dye. The solution volume decline is measured spectrophotometrically.

Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental ...

High-Throughput Assay to Measure Caenorhabditis elegans Food Intake and Lifespan

Quantifying Food Intake in Caenorhabditis elegans by Measuring Bacterial Clearance

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of Caenorhabditis elegans&#160;(C. elegans), an important parameter when studying the genetics of aging or metabolism. In most species, feeding is determined by measuring the difference between the amount of food provided and the amount left after a given time interval. The method presented here uses the same strategy to determine the feeding of C. elegans. It measures the amount of bacteria, the food source of C. elegans, cleared within 72 h. This method uses 96-well microtiter plates and has allowed the screening of hundreds of drugs for their ability to modulate food intake at a speed and depth not possible in other animal models. The strength of this assay is that it allows to measure feeding and lifespan simultaneously and directly measures the disappearance of food and, thus, is based on the same principles used for other organisms, facilitating species-to-species comparison.

Author Spotlight: Exploring the Relationship Between Lipotoxicity and HFpEF 

This protocol describes an assay to quantify Caenorhabditis elegans feeding rate based on measuring the clearance of bacteria in liquid culture.

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

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Neuroscience

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

A Rapid Food-Preference Assay in Drosophila

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate their food environment. The fruit fly, Drosophila melanogaster, is a genetically tractable model organism that is widely used to decipher the molecular, cellular, and neural underpinnings of food preference. To analyze fly food preference, a robust feeding method is needed. Described here is a two-choice feeding assay, which is rigorous, cost-saving, and fast. The assay is Petri-dish-based and involves the addition of two different foods supplemented with blue or red dye to the two halves of the dish. Then, ~70 prestarved, 2-4-day-old flies are placed in the dish and&#160;allowed to choose between blue and red foods in the dark for about 90 min. Examination of the abdomen of each fly is followed by the calculation of the preference index. In contrast to multiwell plates, each Petri dish takes only ~20 s to fill and saves time and effort. This feeding assay can be employed to quickly determine whether flies like or dislike a particular food.

A Rapid Food-Preference Assay in Drosophila

We present a protocol for a two-choice feeding assay for flies. This feeding assay is fast and easy to run and is suitable not only for small-scale laboratory research, but also for high-throughput behavioral screens in flies.

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

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

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on host physiology. It also fluoresces in blue when dying, providing a convenient means of pinpointing death. This property has been exploited to develop high-throughput label-free C. elegans survival assays (LFASS). These involve time-lapse fluorescence recording of worm populations set in multiwell plates, from which population median time of death can be derived. The present study adopts the LFASS approach to screen multiple microbial isolates at once for the effects on C. elegans susceptibility to severe heat and oxidative stresses. Such microbial screening pipeline, which can notably be used to prescreen probiotics, using severe stress resistance as a proxy for host health is reported here. The protocol describes how to grow both C. elegans gut microbiota isolate collections and synchronous worm populations in multiwell arrays before combining them for the assays. The example provided covers the testing of 47 bacterial isolates and one control strain on two worm strains, in two stress assays in parallel. However, the approach pipeline is readily scalable and applicable to the screening of many other modalities. Thus, it provides a versatile setup to rapidly survey a multiparametric landscape of biological and biochemical conditions that impact C. elegans health.

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

Gut microbes may positively or negatively impact the health of their host via specific or conserved mechanisms. Caenorhabditis elegans is a convenient platform to screen for such microbes. The present protocol describes high-throughput screening of 48 bacterial isolates for impact on nematode stress resistance, used as a proxy for worm health.

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on ...

High Throughput Danio Rerio Energy Expenditure Assay

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High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

Quantifying Food Intake in Caenorhabditis elegans by Measuring Bacterial Clearance

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

A Rapid Food-Preference Assay in Drosophila

Measurement of Metabolic Rate in Drosophila using Respirometry

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health