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

1. Collect Eggs from Flies with Genotypes of Interest
NOTE: Ideal crosses consist of 150 virgin flies and at least 75 males. Stock collections should consist of at least 200 flies.
<ol>
	<li>Prepare grape plates.
	<ol>
		<li>Add 37.5 g agar to 1.5 L distilled water in a 4 L flask.</li>
		<li>Autoclave water/agar mix for 50 min at 121 &#186;C (250 &#186;F).</li>
		<li>While water/agar mix is autoclaving, add 50 g sucrose to 500 ml grape juice and stir with a stir bar on a heated stir plate until sucrose is dissolved.</li>
		<li>Turn off the heat and continue stirring to cool the grape juice/sucrose mixture to below 70 &#186;C. Test the internal temperature with an alcohol thermometer.</li>
		<li>Add 3 g Drosophila anti-fungal agent (e.g., Tegosept) to warm grape juice/sucrose mixture and stir to dissolve.</li>
		<li>When water/agar mixture is out of the autoclave, allow the flask to cool to the touch by swirling occasionally.</li>
		<li>Combine water/agar mixture and grape juice/sucrose mixture and stir until well mixed.</li>
		<li>Use a serological pipette to add 8 - 10 ml of the grape mixture to the lid of a small petri plate (35 x 10 mm style). Allow the mixture to crown higher than the height of the lid walls to form a convex shape.</li>
		<li>Allow grape plates to cool for 2 hr at room temperature and then store in an airtight container at 4 &#186;C.</li>
	</ol>
	</li>
	<li>Prepare yeast paste.
	<ol>
		<li>Add 6 ml phosphate buffer solution (PBS) to 4 g live active dried yeast in a 50 ml conical tube and mix with a spatula until smooth.</li>
		<li>Store yeast paste at 4 &#186;C.</li>
	</ol>
	</li>
	<li>Add a small dollop (approximately 1/8 tsp) of yeast paste to the middle of a grape plate. Smooth down any rough edges with a spatula.</li>
	<li>Place grape plates in an incubator until they have reached ambient temperature.</li>
	<li>Transfer flies of interest into an egg collection chamber and place grape plate with yeast paste facing inwards on top. Tape grape plate to the egg collection chamber to avoid letting the plate fall out. Make sure to write important genotype and date information on the bottom of the grape plate.</li>
	<li>Upend the egg collection chamber to allow the flies to lay eggs on the grape plate.</li>
	<li>Place a box or cover over the egg collection chambers and place them in an incubator at 25 &#186;C.</li>
	<li>Allow flies to lay eggs for 4 - 6 hr.</li>
	<li>End collection by taking the grape plate out of the egg collection chamber and transferring the flies back into their bottle of food. Use a spatula to wipe off any excess yeast paste from the grape plate. Store grape plate in a larger petri dish in a humidified incubator at 25 &#186;C for ~ 24 hr.</li>
</ol>
2. Transfer Larvae to Experimental Food
<ol>
	<li>Prepare experimental food. 
	NOTE: This food recipe is based on the Bloomington stock center recipe18. Important changes include increased amount of yeast to optimize the nutritional value of the food as assessed by timing of wandering stage (between 112 - 120 hr after egg collection at 25 &#186;C). While we have found this recipe ideal for the health and development of our experimental larvae, any food recipe is acceptable given calibration with positive and negative controls, examples of which are listed in step 3.10.
	<ol>
		<li>Measure 1,600 ml distilled water.</li>
		<li>Mix 134 g cornmeal, 10.6 g Drosophila Agar Type II, and some of the water (~ 500 ml) in a 1 L beaker. Blend for 2 min with a motorized hand mixer.</li>
		<li>Mix 70 g live active dry yeast, 18.4 g soy flour, 84.8 g malt extract, and some of the water (~ 500 ml) in another 1 L beaker. Blend for 2 min with a motorized hand mixer.</li>
		<li>Swirl each beaker and pour into a 4 L flask. Use remaining water to rinse out the beakers and add into the flask.</li>
		<li>Measure 140 ml light corn syrup and add to the flask, mix by swirling.</li>
		<li>Autoclave for 50 min at 121 &#186;C (250 &#186;F) in the liquid setting.</li>
		<li>After autoclaving, pour food into a 4 L beaker and allow to cool. Help the cooling process by blending with a motorized hand mixer.</li>
		<li>Once the flask has cooled enough to touch, add 8.8 ml propionic acid and 16.8 ml Drosophila anti-fungal agent/ethanol solution. Mix well. (Make Drosophila anti-fungal agent solution by adding 380 g Drosophila anti-fungal agent to 1 L 200 proof ethanol and stirring on a warm plate until dissolved, making sure the solution does not exceed 70 &#186;C.)</li>
		<li>Pour food into vials until the food is approximately 1 inch deep and allow to cool at RT for 2 - 3 hr. Plug vials and store in an incubator at 25 &#186;C. Use within a week.</li>
	</ol>
	</li>
	<li>Plunge a spatula several times into the top layer of the vial of food to score it. This will help the larvae to burrow and feed.</li>
	<li>22 - 24 hr after egg collection has ended, use a small paintbrush to collect 50 first instar (L1) larvae of the same approximate size. Place larvae in the experimental food. Clean the paintbrush on a laboratory wipe and ensure that there are no remaining larvae on the paintbrush before continuing to another genotype.</li>
	<li>Place vial of larvae in an incubator at 25 &#186;C and allow to develop.</li>
</ol>
3. Determine Fat Levels in Larvae
<ol>
	<li>Prepare solutions.
	<ol>
		<li>Prepare 1 L 10x PBS stock.
		<ol>
			<li>Add 80 g NaCl, 2 g KCl, 14.4 g Na2HPO4, and 2.4 g KH2PO4 to 800 ml water in a 2 L beaker containing a stir bar. Stir with no heat until all salts are incorporated. Bring pH to 7.4. Increase volume to 1 L with water.</li>
		</ol>
		</li>
		<li>Prepare 2 L 1x PBS. Add 200 ml 10x PBS stock solution to 1,800 ml water.</li>
		<li>Use this stock of 1x PBS to make 20% w/v sucrose.</li>
	</ol>
	</li>
	<li>Remove the vial of larvae from the incubator once there are 20 - 40 larvae wandering up the sides of the vial (generally on the fifth day after egg collections). Record the date and time of the assay as well as the number of wandering larvae.</li>
	<li>Prepare experimental solution by adding 11.5 ml PBS and 9 ml 20% sucrose to a 50 ml conical tube.</li>
	<li>Add 20% sucrose to the vial until it is 0.5 - 1 inch from the top of the vial.</li>
	<li>Use a spatula to gently stir up the top layer of food to free larvae that are still burrowing in the food. All larvae will rise to the surface of the sucrose solution.</li>
	<li>Use forceps to gently transfer the larvae into the initial sucrose concentration from step 3.3.</li>
	<li>Use a spatula to gently stir the larvae in this solution.</li>
	<li>Cap the conical tube and upend several times to thoroughly mix the solution.</li>
	<li>Uncap the conical tube and swirl to create a gentle vortex.</li>
	<li>Allow the larvae to settle, either floating to the top of the solution or sinking to the bottom. Wait 2 - 5 min for larvae to settle. 
	NOTE: If there are still larvae that are not definitely either floating or sinking, pick a line to use as a cutoff point to label the larvae as either a floater or a sinker. Apply this same criterion to all genotypes. We use the angle at the bottom of the conical tube as our boundary. adp or sir217 mutant larvae may be used as a positive control and lsd219 mutants may be used as a negative control for calibration of the assay.</li>
	<li>Record the number of floating larvae and the specific concentration tested.</li>
	<li>Add 1 ml 20% sucrose to the experimental solution to increase its density.</li>
	<li>Repeat steps 3.7 to 3.10. Record the number of floating larvae and this new concentration.</li>
	<li>Continue steps 3.12 and 3.13 until at least 95% of the larvae are floating.</li>
	<li>Use forceps to collect all larvae into a dissection dish filled with PBS.</li>
	<li>Observe larvae under a microscope and note if there are any small larvae, pre-pupae, pupae, or any other differences between genotypes. 
	NOTE: Ideally, all larvae should appear homogeneous and at the same developmental stage. We have only observed consistent fat measurements under these conditions.</li>
	<li>Record the total number of larvae.</li>
	<li>Using forceps, transfer 10 larvae to a laboratory wipe to dry. Label a microcentrifuge tube and place 10 larvae in the tube.</li>
	<li>Flash freeze the larvae in liquid nitrogen. Store the tube at -20 &#186;C.</li>
</ol>

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	<li>Bier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila,+the+golden+bug,+emerges+as+a+tool+for+human+genetics.">Drosophila, the golden bug, emerges as a tool for human genetics.</a> Nat Rev Genet. 6, 9-23 (2005).</li><li>Liu, Z., Huang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+metabolism+in+Drosophila+development+and+disease+Using+Drosophila+System+to+Study+Lipid+Metabolism+Lipids+Function+in+Drosophila+Early+Development.">Lipid metabolism in Drosophila development and disease Using Drosophila System to Study Lipid Metabolism Lipids Function in Drosophila Early Development.</a> Acta Biochim Biophys Sin. 45 (1), 44-50 (2013).</li><li>Ruden, D. M., Lu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+conservation+of+metabolism+explains+howDrosophila+nutrigenomics+can+help+us+understand+human+nutrigenomics.">Evolutionary conservation of metabolism explains howDrosophila nutrigenomics can help us understand human nutrigenomics.</a> Genes Nutr. 1 (2), 75-84 (2006).</li><li>Wolf, M. J., Rockman, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila,+genetic+screens,+and+cardiac+function.">Drosophila, genetic screens, and cardiac function.</a> Circ. Res. 109, 794-806 (2011).</li><li>Trinh, I., Boulianne, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+obesity+and+its+associated+disorders+in+Drosophila.">Modeling obesity and its associated disorders in Drosophila.</a> Physiology (Bethesda). 28 (2), 117-124 (2013).</li><li>Azeez, O. I., Meintjes, R., Chamunorwa, 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=Fat+body,+fat+pad+and+adipose+tissues+in+invertebrates+and+vertebrates:+the+nexus.">Fat body, fat pad and adipose tissues in invertebrates and vertebrates: the nexus.</a> Lipids Health Dis. 13, 71 (2014).</li><li>Dourlen, P., Sujkowski, A., Wessells, R., Mollereau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+transport+proteins+in+disease:+New+insights+from+invertebrate+models.">Fatty acid transport proteins in disease: New insights from invertebrate models.</a> Prog. Lipid Res. 60, 30-40 (2015).</li><li>Hildebrandt, A., Bickmeyer, I., K&#252;hnlein, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+Drosophila+body+fat+quantification+by+a+coupled+colorimetric+assay.">Reliable Drosophila body fat quantification by a coupled colorimetric assay.</a> PLoS One. 6 (9), e23796 (2011).</li><li>Tennessen, J. M., Barry, W. E., Cox, J., Thummel, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+studying+metabolism+in+Drosophila.">Methods for studying metabolism in Drosophila.</a> Methods. 68, 105-115 (2014).</li><li>Al-Anzi, B., Zinn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+measurement+of+triglycerides+cannot+provide+an+accurate+measure+of+stored+fat+content+in+Drosophila.">Colorimetric measurement of triglycerides cannot provide an accurate measure of stored fat content in Drosophila.</a> PLoS One. 5 (8), e12353 (2010).</li><li>Thanh, N. T. K., Stevenson, G., Obatoml, D., Bach, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Lipids+in+Animal+Tissues+by+High-Performance+Thin-Layer+Chromatography+with+Densitometry.">Determination of Lipids in Animal Tissues by High-Performance Thin-Layer Chromatography with Densitometry.</a> J. Planar Chromatogr. 13, 375-381 (2000).</li><li>Borrull, A., Lopez-Martinez, G., Poblet, M., Cordero-Otero, R., Rozes, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+the+separation+and+quantification+of+neutral+lipid+species+using+GC-MS.">A simple method for the separation and quantification of neutral lipid species using GC-MS.</a> Eur. J. Lipid Sci. 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The authors declare that they have no competing financial interests.

There are a multitude of techniques that have been developed to measure lipid levels8-10. However, each of these methods comes with some major drawbacks that are addressed by the buoyancy-based assay outlined above. First, this assay is extremely quick. Testing a full concentration gradient takes no more than 30 - 60 min. This is a huge improvement on most of the techniques currently in use. For example, lipid quantitation by MS takes 7 - 9 hr to isolate the lipids and another several hou...

Drosophila melanogaster has been used for over a century in the study of genetics and other basic biological questions. In the last few decades, it has become clear that Drosophila is a powerful tool in the study of many human diseases. As 70 - 80% of genes associated with human diseases have an identified fly ortholog1-4, Drosophila provides a simplified yet translatable system in which to study complex diseases. Metabolism in particular has benefited from such study. Not only are the genetics of metabolism well conserved between flies and humans, but the relevant organs and cell types are also very similar2,5. For example, the fat body of the fly stores both triacylglycerides (TAG) and glycogen, functions analogous to those performed in mammalian liver and white adipose tissue6. Using Drosophila as a model for human obesity has vastly improved our understanding of lipid metabolism and the genetics of obesity7. The larval stage of development is particularly useful for studying the segregation of nutrients to storage or utilization as it is dedicated to feeding and the storage of energy to be used during pupariation.
Currently, there are many different quantitative methods of determining fat storage levels in Drosophila. The most widely used method is the coupled colorimetric assay (CCA)8,9. CCA was developed for determining TAG levels in human serum and operates on the premise that glycerol liberated from the backbone of triglycerides will undergo several reactions, ultimately resulting in a redox-coupled reaction generating a colored product. Absorbance of specific wavelengths of light is then measured to determine the initial amount of glycerol present. However, glycerol can also be liberated from mono- and diacylglycerides in addition to TAG and therefore may not be an accurate measure of stored body fat9. Furthermore, eye pigment of crushed adult flies can interfere with some absorbance readings and complicate results9,10. Therefore, CCA must be accompanied and validated by thin layer chromatography (TLC), which allows for the separation of most lipid families that can be quantitated by densitometry10,11. However, some lipid classes like sterols cannot be analyzed and must be quantified a different way12. Mass spectrometry (MS) is an accurate way to quantitate all classes of major cellular lipids12,13. However, the lipid extraction procedures necessary to analyze by MS are both time consuming (most taking nearly a full day) and costly. Here we present an alternative method to quickly and cheaply determine organismal fat levels in the L3 larvae of Drosophila melanogaster.
The method presented below exploits the difference in density between fat tissue and lean tissue. Mammalian fat tissue has a density of approximately 0.9 g/ml14 while skeletal muscle as a density of 1.06 g/ml15. This difference means that animals with higher stores of fat will have lower density than animals with lower stores of fat, which will allow them to float better in a solution of fixed density. This property allows for extremely quick screening of a large number of animals while being both inexpensive and non-invasive. Buoyancy-based analysis has been used both to confirm the phenotypes of altering known regulators of fat levels as well as to identify new genetic and neurological regulators of obesity16,17.

<table border="0" cellpadding="0" cellspacing="0" width="670">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bacto Agar</td>
			<td style="width:168px;">VWR</td>
			<td style="width:119px;">214030</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Welch&#39;s Original 100% Grape Juice</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sucrose</td>
			<td style="width:168px;">Fisher</td>
			<td style="width:119px;">S512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tegosept</td>
			<td style="width:168px;">Genesee Scientific</td>
			<td style="width:119px;">20-259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td>200 proof</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Baker&#39;s yeast</td>
			<td style="width:168px;">Fleichmann&#39;s</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Yellow Corn Meal</td>
			<td style="width:168px;">Quaker</td>
			<td style="width:119px;"></td>
			<td>Enriched degerminated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Drosophila Agar Type II</td>
			<td style="width:168px;">Apex</td>
			<td style="width:119px;">66-104</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Soy Flour</td>
			<td style="width:168px;">ADM Specialty Ingredients</td>
			<td style="width:119px;">062-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Malt Extract</td>
			<td style="width:168px;">Breiss</td>
			<td style="width:119px;">5748</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Light Corn Syrup</td>
			<td style="width:168px;">Karo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Propionic Acid</td>
			<td>VWR</td>
			<td>U330-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
			<td style="width:168px;">Fisher</td>
			<td style="width:119px;">50147491</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium phosphate monobasic</td>
			<td>Sigma</td>
			<td>P5655-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium phosphate anhydrous</td>
			<td style="width:168px;">Fisher</td>
			<td style="width:119px;">S3933</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium chloride</td>
			<td>Fisher</td>
			<td>P330-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 x 10mm petri plate</td>
			<td style="width:168px;">Fisher</td>
			<td>08-757-100A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50mL Conical</td>
			<td style="width:168px;">Fisher</td>
			<td style="width:119px;">50-869-569</td>
			<td></td>
		</tr>
	</tbody>
</table>

a buoyancy-based method of determining fat levels in drosophila

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

Figure 1 represents an example of how the buoyancy-based assay can detect both higher and lower fat stores in genetically manipulated larvae. Figure 1A shows that excitation or silencing of a certain subset of neurons (E347) in the larval brain induces lower and higher fat stores respectively. The same genetic background control was used in both cases. Figure 1B provides an example of how this phenotype obtained by the density assay is co...

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A Buoyancy-based Method of Determining Fat Levels in Drosophila

Here we present a method to measure organismal fat levels in the third instar (L3) larval stage of Drosophila melanogaster. This method exploits the comparatively low density of fat tissue to differentiate between larvae with altered fat stores. Buoyancy-based analysis is a valuable tool for rapid, reproducible, and economical screening.

A Buoyancy-based Method of Determining Fat Levels in Drosophila

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

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Research

JoVE Journal

Biology

8.5K Views.  University of Colorado Anschutz Medical Campus. Here we present a method to measure organismal fat levels in the third instar (L3) larval stage of Drosophila melanogaster. This method exploits the comparatively low density of fat tissue to differentiate between larvae with altered fat stores. Buoyancy-based analysis is a valuable tool for rapid, reproducible, and economical screening.

Video: A Buoyancy-based Method of Determining Fat Levels in Drosophila

Studying Aggression in Drosophila (fruit flies)

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources. This complex social behavior is influenced by genetic, hormonal and environmental factors. In many organisms, aggression is critical to survival but controlling and suppressing aggression in distinct contexts also has become increasingly important. In recent years, invertebrates have become increasingly useful as model systems for investigating the genetic and systems biological basis of complex social behavior. This is in part due to the diverse repertoire of behaviors exhibited by these organisms. In the accompanying video, we outline a method for analyzing aggression in Drosophila whose design encompasses important eco-ethological constraints. Details include steps for: making a fighting chamber; isolating and painting flies; adding flies to the fight chamber; and video taping fights. This approach is currently being used to identify candidate genes important in aggression and in elaborating the neuronal circuitry that underlies the output of aggression and other social behaviors.

Studying Aggression in Drosophila fruit flies

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources.  This complex social behavior is influenced by ...

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 ...

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 ...

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

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

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

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

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

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

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

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

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.

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

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

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

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of genetic manipulations with this model has supplanted pharmacological approaches that are commonplace in other animal models and cell-based assays. Here we describe recent advances in a protocol that enables application of small molecules to the developing fruit fly embryo. The method details steps to overcome the impermeability of the eggshell while maintaining embryo viability. Eggshell permeabilization across a broad range of developmental stages is achieved by application of a previously described d-limonene embryo permeabilization solvent (EPS1) and by aging embryos at reduced temperature (18 &#176;C) prior to treatments. In addition, use of a far-red dye (CY5) as a permeabilization indicator is described, which is compatible with downstream applications involving standard red and green fluorescent dyes in live and fixed preparations. This protocol is applicable to studies using bioactive compounds to probe developmental mechanisms as well as for studies aimed at evaluating teratogenic or pharmacologic activity of uncharacterized small molecules.

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

Introduction of small molecules to the developing Drosophila embryo offers great potential for characterizing biological activity of novel compounds, drugs, and toxins as well as for probing fundamental developmental pathways. Methods described herein outline steps that overcome natural barriers to this approach, expanding the utility of the Drosophila embryo model.

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

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

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

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