This work was supported by National Institutes of Health grants F32 NS078958 (DTB) and R01 AG033620 (BG).

1. Assemble Flight Tester
<ol>
	<li>Secure flight cylinder to Ring Stand 1 using chain clamps. (Leave approximately 3 cm underneath the cylinder for weigh dish.) 
	(Note: The flight cylinder we use is 90 cm long with a diameter of 13.5 cm.)</li>
	<li>Insert weigh dish with a thin layer of mineral oil underneath the flight cylinder.</li>
	<li>Secure funnel to Ring Stand 2 using a ring clamp and claw clamp. Adjust the height of the funnel so that the bottom of the funnel is flush with the top of the flight cylinder. (Note: the tip diameter of the funnel must be less than the outer diameter of vials placed into the drop tubes so that the vials will not fall through.)</li>
	<li>Insert drop tube into the top of the funnel and secure using a claw clamp. 
	(Note: We use a drop tube that is 25 cm long. Dropping fly-containing vials from this height allows consistent ejection of all flies with uniform force. The inner diameter of the drop tube should be slightly larger than the outer diameter of the vial to allow the vial to drop freely.)</li>
	<li>Cut polyacrylamide sheet(s) to the proper size. (Note: To aid in inserting and removing the sheet, the width should be slightly smaller than the inner circumference of the flight cylinder).</li>
	<li>Apply a thin layer of Tangle-Trap to the sheet. Allow to sit for 1 hr before use. (Note: Leave enough room at the top and bottom of the sheet (approximately&#160;3 cm) uncoated to grasp the sheet for insertion/removal.)</li>
	<li>Insert the polyacrylamide sheet in the flight cylinder.</li>
	<li>Assemble the camera track using pine support brackets. (Note: ensure that the bottom of the track can support the camera without blocking the lens. Refer to Figure 1B.)</li>
	<li>Add stoppers and screw into place. (Note: place the stoppers in locations that will allow the camera to view the entire plastic sheet in panoramic mode.)</li>
</ol>
2. Run Experiment
<ol>
	<li>Collect vials of flies to be tested. For best results, use no more than 20 flies/vial.</li>
	<li>Gently tap flies to bottom of vial, unplug, then insert into drop tube and release vial. 
	(Note: The vial falls down the drop tube until it hits the narrow funnel opening. When the vial hits the funnel, the flies are ejected into the flight cylinder.)</li>
	<li>Lift the drop tube to remove the empty vial. 
	(Note: Multiple vials of flies of the same test group can be assayed on a single polyacrylamide sheet. We find that up to 200 flies (10 vials of 20 flies each) can be tested and imaged readily on a single sheet.</li>
	<li>Remove the plastic sheet and place it on a flat white surface. 
	(Note: white poster board may be used if bench tops are dark colored.)</li>
	<li>Assemble the camera track over the plastic sheet. The camera should be sufficiently high above the sheet to have both the top and bottom of the sheet in the field of view.</li>
	<li>Slide the camera along the track while holding the &#34;capture&#34; button to acquire a panoramic image.</li>
	<li>The number of flies landing in the oil may be counted manually for each trial.</li>
	<li>Repeat steps 2.2-2.7 for all conditions in a given experiment. The flies can be removed from the sheet between each trial. Alternatively, several sheets may be used, with a new sheet for each trial.</li>
</ol>
3. Data Collection
<ol>
	<li>Open image files using ImageJ software.</li>
	<li>Crop images if necessary to include only the landing surface area. (This is the area coated in Tangle-Trap.)</li>
	<li>Convert images to 8-bit grayscale.</li>
	<li>Create a &#34;Threshold&#34; to filter out the white background. 
	(Image &#8594; Adjust &#8594; Threshold).</li>
	<li>Set the parameters to identify each fly using the &#34;analyze particles&#34; menu. 
	(Analyze &#8594; Analyze Particles) Define the parameters used to identify a particle. With our set up, we find that using an area of 5-90 pixels2 and a circularity of 0.4-1.0 will accurately identify all samples.</li>
	<li>Measure the location of each fly using the generated list of coordinates for each particle. The x-coordinate in pixels can be converted to centimeters to calculate the landing height.</li>
	<li>Import table into a spreadsheet (such as Microsoft Excel).</li>
</ol>

<ol>
	<li>Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+behavior.">Genetic dissection of behavior.</a> Sci. Am. 229, 24-37 (1973).</li><li>Ali, Y. O., Escala, W., Ruan, K., Zhai, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+locomotor,+learning,+and+memory+deficits+in+Drosophila+models+of+neurodegeneration.">Assaying locomotor, learning, and memory deficits in Drosophila models of neurodegeneration.</a> J. Vis. Exp. , e2504 (2011).</li><li>de Vries, S. E., Clandinin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+Stimulation+of+Escape+Behavior+in+Drosophila+melanogaster.">Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster.</a> J. Vis. Exp. , e50192 (2013).</li><li>Mundiyanapurath, S., Certel, S., Kravitz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+aggression+in+Drosophila+(fruit+flies).">Studying aggression in Drosophila (fruit flies).</a> J. Vis. Exp. , e155 (2007).</li><li>Nichols, C. D., Becnel, J., Pandey, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+assay+Drosophila+behavior.">Methods to assay Drosophila behavior.</a> J. Vis. Exp. , e3795 (2012).</li><li>Shiraiwa, T., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proboscis+extension+response+(PER)+assay+in+Drosophila.">Proboscis extension response (PER) assay in Drosophila.</a> J. Vis. Exp. , e193 (2007).</li><li>Lloyd, T. E., Taylor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flightless+flies:+Drosophila+models+of+neuromuscular+disease.">Flightless flies: Drosophila models of neuromuscular disease.</a> Ann. N.Y. Acad. Sci. 1184, e1-e20 (2010).</li><li>Atkinson, N. S., 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=Molecular+separation+of+two+behavioral+phenotypes+by+a+mutation+affecting+the+promoters+of+a+Ca-activated+K+channel.">Molecular separation of two behavioral phenotypes by a mutation affecting the promoters of a Ca-activated K channel.</a> J. Neurosci. 20, 2988-2993 (2000).</li><li>Atkinson, N. S., Robertson, G. A., Ganetzky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+component+of+calcium-activated+potassium+channels+encoded+by+the+Drosophila+slo+locus.">A component of calcium-activated potassium channels encoded by the Drosophila slo locus.</a> Science. 253, 551-555 (1991).</li><li>Elkins, T., Ganetzky, B., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+mutation+that+eliminates+a+calcium-dependent+potassium+current.">A Drosophila mutation that eliminates a calcium-dependent potassium current.</a> Proc. Natl. Acad. Sci. U.S.A. 83, 8415-8419 (1986).</li><li>Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Exp. Gerontol. 40, 386-395 (2005).</li><li>Fry, S. N., Rohrseitz, N., Straw, A. D., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+control+of+flight+speed+in+Drosophila+melanogaster.">Visual control of flight speed in Drosophila melanogaster.</a> J. Exp. Biol. 212, 1120-1130 (2009).</li><li>Mronz, M., Lehmann, F. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+free-flight+response+of+Drosophila+to+motion+of+the+visual+environment.">The free-flight response of Drosophila to motion of the visual environment.</a> J. Exp. Biol. 211, 2026-2045 (2008).</li></ol>

The authors have no conflicts of interest to disclose.

Using the methods described here, we have been able to rapidly assess flight performance of a large number of Drosophila mutants, providing greater efficiency than previously possible. For our experiments, we routinely separate males and females and raise them at low density (less than 20 flies/vial) to limit aggression that could damage wings. Another important consideration is to control properly for differences in flight performance owing to differences in genetic background. We also find it helps to allow fl...

Drosophila has long been used to study the genetic basis of behavior1, and researchers have devised a number of ways to analyze various types of behavior2-6. Flies have been particularly useful in providing useful models of neuromuscular disorders7. A common assay used to study locomotor behavior is flight performance. The original flight tester is useful for identifying flight defective mutants and for quantitative assessment of flight ability1, but it has several shortcomings that limit its application for high throughput screens: the use of oil-coated cylinders is messy and cumbersome, certain features such as the length of the cylinder and introduction of flies into the tube with variable force reduce quantitative accuracy; and it is difficult to recover live flies from the tester. To overcome these limitations, we have modified the flight tester to include a number of improvements. We added a &#34;drop tube&#34; to introduce flies to eliminate variability between experiments and users. We use removable acrylic sheets coated with an adhesive that allows for easier cleanup and recovery of individual flies. We have increased the length of the flight tube to improve quantitative accuracy and reliability. Finally, we use a digital camera and imaging software to calculate the landing heights of flies. We believe these improvements will be useful to any laboratory interested in conducting large-scale genetic screens for defects in flight performance.

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			<td height="33" style="height:33px;width:216px;">white poster board</td>
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			<td height="33" style="height:33px;width:216px;">polystyrene weighing dish</td>
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			<td height="33" style="height:33px;width:216px;">ImageJ Software</td>
			<td style="width:171px;">National Institutes of Health</td>
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			<td>http://rsb.info.nih.gov/ij/</td>
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			<td height="33" style="height:33px;width:216px;">digital camera</td>
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			<td height="33" style="height:33px;width:216px;">polycarbonate cylinder (drop tube)</td>
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			<td height="33" style="height:33px;width:216px;">flight cylinder (acrylic)</td>
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			<td height="33" style="height:33px;width:216px;">ring stand (x2)</td>
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			<td height="33" style="height:33px;width:216px;">glass vials</td>
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</table>

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.

Figure 1A shows a schematic of the updated flight tester assembly. Figure 1B illustrates the track design allow the camera to take a panoramic image without blocking the field of view. Representative results are shown in Figure 2, where the flight performance of slowpoke mutant flies, which have a known flight defect8-10, are compared to wild-type Canton-S flies. Control flies consistently land near the top of the cylinder, with very little s...

Watch this Scientific Journal Video about An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila at JoVE.com

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.

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 ...

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11.7K Aufrufe.  University of Wisconsin-Madison.  Hier beschreiben wir ein Verfahren zum schnellen und genauen Messung der Flugleistung in Drosophila, was eine Hochdurchsatz-Screening. 

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

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

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

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Also, the recent deluge of research on the neurophysiology and neurobehavioral genetics of olfaction in Drosophila has motivated ever more technically sophisticated and quantitative behavioral assays. Here, we modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. A fly is glued to a small steel pin and suspended in a magnetic field that enables it to yaw freely. Small diameter food odor plumes are directed downward over the fly s head, eliciting stable tracking by a hungry fly. Here we focus on the critical mechanics of tethering, aligning the magnets, devising the odor plume, and confirming stable odor tracking.

Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

Visually Mediated Odor Tracking During Flight in 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 stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

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 ...

An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high concentrations of phenolic compounds and polysaccharides that interfere with RNA purification. Isolation of high-quality RNA from these species requires rigorous tissue collection procedures in the field and the employment of an RNA isolation protocol comprised of multiple organic extraction steps in order to isolate RNA of sufficient quality for microarray and other genomic analyses. The isolation of high-quality RNA from field-collected loblolly pine samples can be challenging, but several modifications to standard tissue and RNA isolation procedures greatly improve results. The extent of general RNA degradation increases if samples are not properly collected and transported from the field, especially during large-scale harvests. Total RNA yields can be increased significantly by pulverizing samples in a liquid nitrogen freezer mill prior to RNA isolation, especially when samples come from woody tissues. This is primarily due to the presence of oxidizing agents, such as phenolic compounds, and polysaccharides that are both present at high levels in extracts from the woody tissues of most conifer species. If not removed, these contaminants can carry over leading to problems, such as RNA degradation, that result in low yields and a poor quality RNA sample. Carryover of phenolic compounds, as well as polysaccharides, can also reduce or even completely eliminate the activity of reverse transcriptase or other polymerases commonly used for cDNA synthesis. In particular, RNA destined to be used as template for double-stranded cDNA synthesis in the generation of cDNA libraries, single-stranded cDNA synthesis for PCR or qPCR's, or for the synthesis of microarray target materials must be of the highest quality if researchers expect to obtain optimal results. RNA isolation techniques commonly employed for many other plant species are often insufficient in their ability to remove these contaminants from conifer samples and thus do not yield total RNA samples suitable for downstream manipulations. In this video we demonstrate methods for field collection of conifer tissues, beginning with the felling of a forty year-old tree, to the harvesting of phloem, secondary xylem, and reaction wood xylem. We also demonstrate an RNA isolation protocol that has consistently yielded high-quality RNA for subsequent enzymatic manipulations.

An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species

Many plant tissues, including phloem and xylem from loblolly pine (Pinus taeda L.), contain high levels of phenolics and polysaccharides that interfere with RNA purification. This presentation discusses techniques for the harvest of field-grown tissues and isolation of RNA of sufficient quality for microarrays and other genomic analyses.

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high ...

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders such as diabetes and cardiovascular disease 1-3. An important topic of research into these associated health problems involves the role of endurance exercise as a beneficial intervention.
Exercise training is an inexpensive, non-invasive intervention with several beneficial results, including reduction in excess body fat 4, increased insulin sensitivity in skeletal muscle 5, increased anti-inflammatory and antioxidative responses 6, and improved contractile capacity in cardiomyocytes 7. Low intensity exercise is known to increase mitochondrial activity and biogenesis in humans 8 and mice, with the transcriptional coactivator PGC1-&alpha; as an important intermediate 9,10. 
Despite the importance of exercise as a tool for combating several important age-related diseases, extensive longitudinal genetic studies have been impeded by the lack of an endurance training protocol for a short-lived genetic model species. The variety of genetic tools available for use with Drosophila, together with its short lifespan and inexpensive maintenance, make it an appealing model for further study of these genetic mechanisms. With this in mind we have developed a novel apparatus, known as the Power Tower, for large scale exercise-training in Drosophila melanogaster 11. The Power Tower utilizes the flies' instinctive negative geotaxis behavior to repetitively induce rapid climbing. Each time the machine lifts, then drops, the platform of flies, the flies are induced to climb. Flies continue to respond as long as the machine is in operation or until they become too fatigued to respond. Thus, the researcher can use this machine to provide simultaneous training to large numbers of age-matched and genetically identical flies. Additionally, we describe associated assays useful to track longitudinal progress of fly cohorts during training.

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

We describe the first endurance training protocol for an important genetic model species, Drosophila melanogaster, and outline several assays to chart improvements in mobility following training.

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders ...

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.

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

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

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 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.

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

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...