Special thanks to the University of Missouri-Kansas City and the Jeffrey L. Price laboratory.

1. Fly food production
<ol>
	<li>Per every 1 L of water, prepare fly food consisting of 4.69 g of dried molasses, 19.70 g of dry active yeast, 87.22 g of corn meal and 7.83 g of agar.</li>
	<li>Combine the contents listed above in a crockpot and turn the heat to 250 &#176;F. Mix well as ingredients are added.</li>
	<li>Keep the lid on the crockpot as the fly food is heating while also mixing the contents every 10 min until it reaches a rolling boil. Allow the rolling boil to continue for 20 min before turning off the heat.</li>
	<li>Add 83.60 mL of water and keep the lid of the crockpot off as the food cools.</li>
	<li>Mix and record the temperature of the food every 10 min with a glass thermometer. Avoid letting a layer of film settle on top of the food.</li>
	<li>Once the food has cooled to 60 &#176;C, add 10.44 mL of Tegosept and 5.51 mL of propionic acid per liter of water added (see step 1.1).</li>
	<li>Mix well and turn heat up to 60 &#176;C to prevent the food from becoming too cool.</li>
	<li>Pump the fly food as needed using the Droso-filler.</li>
	<li>For narrow vials, pump 10 mL and for 6 oz square bottom bottles pump 60 mL.</li>
</ol>
2. Collecting flies of defined age
<ol>
	<li>Monitor bottles of wild type flies stored at 25 &#176;C for 5-7 days until large amounts of pupa (about 200 pupa) are attached to the side of the bottle. The bottles used are 6 oz Drosophila stock bottles with square bottoms.</li>
	<li>Clear existing adults from the bottle. Either tip the adults into a new bottle or place them in 70% ethanol. Use the dull end of a #0 paintbrush to push any remaining adults into the fly food. Be sure to wipe the paint brush down with 70% ethanol before and after uses.</li>
	<li>Allow cleared bottles to sit for 3 days in a 25 &#176;C incubator to allow for the next generation to eclose. These flies will be between 0 and 3 days old.</li>
</ol>
3. Fly separation
<ol>
	<li>After 3 days, separate males from females and collect the desired number for each sex for each of the four time points. Flies can be differentiated by sex by examining genitalia; males have dark rounded genitalia whereas females have lighter, more pointed genitalia. Females are also much larger than male flies.
	<ol>
		<li>Use a CO2 anesthesia pad to effectively separate the flies and differentiate between the sexes. Move the flies with paint brushes to avoid killing them.</li>
		<li>Collect 100 males and 100 females for each time point, with 50 males per vial and 100 females per vial. Males tend to be more aggressive and their social interactions lead to a lot of deaths when there are 100 individuals in a vial. The females-up to 100 individuals- are unaffected while contained in the vial.</li>
	</ol>
	</li>
	<li>Perform the collections at the following time points: ZT1, ZT7, ZT13 and ZT19 (Table 1). Note that collections done in the dark (ZT12-ZT24) are light sensitive whereas ZT0-ZT11 collections are done during lights-on times and room lights can be on. Please note that two separate incubators are used with inverse 12 hour light patterns to allow for all collections to occur during the day and not overnight.</li>
	<li>Use excess flies to create new bottles of flies for future entrainment. Place 25 females with 5-7 males in each new bottle and place in the incubator at 25 &#176;C.</li>
</ol>
4. Fly incubation
<ol>
	<li>Allow the flies to stay in light regulated incubators for 3-5 days to allow circadian entrainment to occur. Ensure that incubators are light-tight because even small amounts of light pollution will disturb entrainment.</li>
</ol>
5. Immunofluorescence fixation
<ol>
	<li>After entrainment, prepare new vials of fixation solution for samples that will be used for immunofluorescence. Prior to removing the flies from the incubator, add 4.8 mL of 4% formaldehyde diluted in 1x PBS + 0.1% Tween-20 to each new narrow vial for every 100 flies that are to be fixated. Each vial will house 100 male or 100 female Circadian entrained flies. Place the narrow vials in ice.</li>
</ol>
6. Immunofluorescence collection
<ol>
	<li>When collecting the flies from the incubator for immunofluorescence, remove the bottle cap and quickly invert the bottle into the funnel. Gently tap the flies into the solution via the funnel to help guide the flies into the vial; combine two tubes of the 50 males for a total of 100 males in one vial and use a single tube of 100 females for the other vial.</li>
	<li>Perform ZT13 and ZT19 collections in the dark; use a red light in order to see as drosophila are far less sensitive to these light wavelengths and are therefore less prone to light pollution13. Cryptochrome protein, in particular, is especially sensitive to blue light, which must be avoided14.
	<ol>
		<li>To reduce light exposure, ensure the room of collection is light tight with any sources of light blocked out or covered. Wrap these vials in foil so that ZT13 and ZT19 flies are not exposed to light when placed on the nutating mixer in the following step. ZT1 and ZT7 flies are not light sensitive and can be placed on the mixer without foil covering.</li>
	</ol>
	</li>
</ol>
7. Nutating mixer and storage
<ol>
	<li>After the flies have been collected, tape the top of the vials containing fixative to avoid spillage and place them on the nutating mixer at 165 RPM at 4 &#176;C for 4 h. The flies are no longer light sensitive after this fixation step, so foil may be removed to verify the solution is moving and flies are being submerged in the fixative.</li>
	<li>After removing the fly containing vials from the nutating mixer remove the formaldehyde and wash three times with 3,000 &#181;L of 1x PBS, inverting the vial with each wash.</li>
	<li>Store the vials bearing immunofluorescence samples at 4 &#176;C to await future immunofluorescence15.</li>
</ol>
8. Collection for protein extraction
<ol>
	<li>Prepare four 50 mL tubes and a Dewar containing liquid nitrogen for preservation of samples for protein extraction</li>
	<li>For collecting flies for protein or nucleic acid extraction, transfer flies from the bottles to the tube in the same manner as in step 6.1 and quickly cap the tube to prevent the release of flies and place the tube into liquid nitrogen to snap freeze.</li>
</ol>
9. Storage for protein extraction
<ol>
	<li>Store snap frozen samples for protein extraction at -80 &#176;C. These can be processed according to the protein extraction protocol appropriate for downstream analysis, including immunoblotting16.</li>
</ol>

<ol>
	<li>Halberg, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+physiological+and+clinical+aspects+of+24-hour+periodicity.">Some physiological and clinical aspects of 24-hour periodicity.</a> The Journal-Lancet. 73 (1), 20-32 (1953).</li><li>Smarr, B. L., Jennings, K. J., Driscoll, J. R., Kriegsfeld, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+time+to+remember:+the+role+of+circadian+clocks+in+learning+and+memory.">A time to remember: the role of circadian clocks in learning and memory.</a> Behavioral Neuroscience. 128 (3), 283-303 (2014).</li><li>Leng, Y., Musiek, E. S., Hu, K., Cappuccio, F. P., Yaffe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+between+circadian+rhythms+and+neurodegenerative+diseases.">Association between circadian rhythms and neurodegenerative diseases.</a> The Lancet. Neurology. 18 (3), 307-318 (2019).</li><li>Hood, S., Amir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodegeneration+and+the+Circadian+Clock.">Neurodegeneration and the Circadian Clock.</a> Frontiers in aging Neuroscience. 9, 170 (2017).</li><li>Sulli, G., Lam, M. T. Y., Panda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+Circadian+Clock+and+Cancer:+New+Frontiers+for+Cancer+Treatment.">Interplay between Circadian Clock and Cancer: New Frontiers for Cancer Treatment.</a> Trends in Cancer. 5 (8), 475-494 (2019).</li><li>Zehring, W. A., 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=P-element+transformation+with+period+locus+DNA+restores+rhythmicity+to+mutant,+arrhythmic+Drosophila+melanogaster.">P-element transformation with period locus DNA restores rhythmicity to mutant, arrhythmic Drosophila melanogaster.</a> Cell. 39 (2 Pt 1), 369-376 (1984).</li><li>Bargiello, T. A., Jackson, F. R., Young, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+circadian+behavioural+rhythms+by+gene+transfer+in+Drosophila.">Restoration of circadian behavioural rhythms by gene transfer in Drosophila.</a> Nature. 312 (5996), 752-754 (1984).</li><li>Siwicki, K. K., Eastman, C., Petersen, G., Rosbash, M., Hall, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+to+the+period+gene+product+of+Drosophila+reveal+diverse+tissue+distribution+and+rhythmic+changes+in+the+visual+system.">Antibodies to the period gene product of Drosophila reveal diverse tissue distribution and rhythmic changes in the visual system.</a> Neuron. 1 (2), 141-150 (1988).</li><li>Hardin, P. E., Hall, J. C., Rosbash, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feedback+of+the+Drosophila+period+gene+product+on+circadian+cycling+of+its+messenger+RNA+levels.">Feedback of the Drosophila period gene product on circadian cycling of its messenger RNA levels.</a> Nature. 343 (6258), 536-540 (1990).</li><li>Liu, X., 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=The+period+gene+encodes+a+predominantly+nuclear+protein+in+adult+Drosophila.">The period gene encodes a predominantly nuclear protein in adult Drosophila.</a> Journal of Neuroscience. 12 (7), 2735-2744 (1992).</li><li>Vosshall, L. B., Price, J. L., Sehgal, A., Saez, L., Young, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Block+in+nuclear+localization+of+period+protein+by+a+second+clock+mutation,+timeless.">Block in nuclear localization of period protein by a second clock mutation, timeless.</a> Science (New York, N.Y). 263 (5153), 1606-1609 (1994).</li><li>Price, J. L., 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=double-time+is+a+novel+Drosophila+clock+gene+that+regulates+period+protein+accumulation.">double-time is a novel Drosophila clock gene that regulates period protein accumulation.</a> Cell. 94 (1), 83-95 (1998).</li><li>Hanai, S., Hamasaka, Y., Ishida, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+entrainment+to+red+light+in+Drosophila:+requirement+of+Rhodopsin+1+and+Rhodopsin+6.">Circadian entrainment to red light in Drosophila: requirement of Rhodopsin 1 and Rhodopsin 6.</a> Neuroreport. 19 (14), 1441-1444 (2008).</li><li>Vinayak, P., 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=Exquisite+light+sensitivity+of+Drosophila+melanogaster+cryptochrome.">Exquisite light sensitivity of Drosophila melanogaster cryptochrome.</a> PLoS Genetics. 9 (7), e1003615 (2013).</li><li>Kelly, S. M., Elchert, A., Kahl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+and+Immunofluorescent+Staining+of+Mushroom+Body+and+Photoreceptor+Neurons+in+Adult+Drosophila+melanogaster+Brains.">Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains.</a> Journal of Visualized Experiments. (129), e56174 (2017).</li><li>Au-Eslami, A., Au-Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+Blotting:+Sample+Preparation+to+Detection.">Western Blotting: Sample Preparation to Detection.</a> Journal of Visualized Experiments. (44), e2359 (2010).</li><li>Fan, J. Y., Muskus, M. J., Price, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entrainment+of+the+Drosophila+circadian+clock:+more+heat+than+light.">Entrainment of the Drosophila circadian clock: more heat than light.</a> Science's signal Transduction Knowledge Environment. (413), pe65 (2007).</li><li>Miyasako, Y., Umezaki, Y., Tomioka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separate+sets+of+cerebral+clock+neurons+are+responsible+for+light+and+temperature+entrainment+of+Drosophila+circadian+locomotor+rhythms.">Separate sets of cerebral clock neurons are responsible for light and temperature entrainment of Drosophila circadian locomotor rhythms.</a> Journal of Biological Rhythms. 22 (2), 115-126 (2007).</li><li>Edery, I., Zwiebel, L. J., Dembinska, M. E., Rosbash, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+phosphorylation+of+the+Drosophila+period+protein.">Temporal phosphorylation of the Drosophila period protein.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (6), 2260-2264 (1994).</li><li>Helfrich-Forster, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuroarchitecture+of+the+circadian+clock+in+the+brain+of+Drosophila+melanogaster.">The neuroarchitecture of the circadian clock in the brain of Drosophila melanogaster.</a> Microscopy Research and Technique. 62 (2), 94-102 (2003).</li><li>Price, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+screens+for+clock+mutants+in+Drosophila.">Genetic screens for clock mutants in Drosophila.</a> Methods in Enzymology. 393, 35-60 (2005).</li></ol>

Researchers utilize this entrainment protocol with success and consistency. This procedure allows the fixation of a large sampling pool that can be stored for future analysis. Additionally, this strategy preserves the neurological patterns induced by entrainment for future examination.
Fixation for storage is a major component of the entrainment process as it helps to stabilize brain tissue and it allows for more time to analyze each brain from the data pool thus minimizing waste from brains that lose viability due to age21. The main goal is to circadian entrain as many flies as possible so that there is continuous inventory available for head dissections and ultimately immunofluorescence or protein extraction to observe the findings and determine if results are of high confidence. To ensure that circadian entrainment is preserved through fixation, it is integral that any source of light pollution is eliminated. The fixation process allows for Drosophila to be stored while maintaining its neurological &#34;timestamp&#34; so that they can be dissected later and analyzed with no noticeable differences to flies that are dissected and have undergone immunofluorescence immediately after entrainment. For the purposes of fixation prior to immunofluorescence, the lab has determined with consistency that flies are viable at least up to 1 month. Fixations for western blot protein extraction render the brains viable indefinitely when stored at -80 &#176;C.
Another critical protocol step is the sexing of the flies. It is important that this step is done accurately as having both sexes in the same vial prior to fixation can lead to mating, which will yield new flies that are of younger age and corrupt protein analysis if males are accidentally examined instead of females or vice versa. Additionally, when sexing it is important to remove larvae specimens that are at times attached to females. This prevents the development of new progeny inside the female vial that could potentially corrupt results.
The next step for the entrainment protocol may be with items related to data analysis. The focus of the protocol is protein localization, but if there are other variables that are impacted by circadian entrainment, they must be explored through new avenues, often requiring protein or nucleic acid extraction. Additionally, there are other proteins of the brain that may still be analyzed via this protocol. The experiments associated with the protocol analyzed certain proteins but the list of genes and proteins that play a role in circadian biology has not been exhausted. The protocol is effective in accomplishing the goal of establishing a circadian rhythm, however, the applications are wide-ranging.

Almost all organisms on Earth, from the largest down to single celled, have an internal biological clock with a cycle of about one day. This is known as the Circadian rhythm (coined in 1953 by Franz Halberg from the Latin terms circa = about/approximately and &#34;dies&#34; = day)1. Although components of the core clock are known and their rudimentary mechanisms of function conceptualized, there is still much to understand about how biological rhythms are maintained throughout the body. Importantly, misregulation of biological rhythms is associated with poor health outcomes including poor memory formation, sleep disorders, seasonal affect disorder, depression, bipolar disorder, diabetes, obesity, neurodegeneration, and cancer2,3,4,5.
Drosophila is a well-established model for investigation of circadian biology. Genetically and biochemically tractable, large numbers are easily entrained (as will be shown). In fact, all seven publications cited as key publications of support in awarding the Nobel Prize for the discovery of circadian rhythms leveraged these strengths of the Drosophila model6,7,8,9,10,11,12.
Additionally, we show effective strategies for collecting entrained flies for the purposes of either immunofluorescence, nucleic acid, or protein extraction-based analysis. Using these strategies, one may process and store larger amounts of samples for analysis in the future. These methods are very advantageous in that they are reproducible and can yield hundreds of entrained flies that can be a part of a large data pool.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100-1000uL pipette</td>
			<td>Eppendorf</td>
			<td>ES-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>10-100uL pipette</td>
			<td>Eppendorf</td>
			<td>ES-100</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde Solution</td>
			<td>15710</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1X PBS</td>
			<td>Caisson Labs</td>
			<td>PBL01-6X100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher Scientific</td>
			<td>BP1423500</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia Filter Connection Kit</td>
			<td>World Precision Instruments</td>
			<td>EZ-251A</td>
			<td></td>
		</tr>
		<tr>
			<td>Corn meal</td>
			<td>Genesee Scientific</td>
			<td>62-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dried Molasses</td>
			<td>Food Service Direct</td>
			<td>OT280504</td>
			<td></td>
		</tr>
		<tr>
			<td>Droso-filler Food Pump</td>
			<td>geneseesci.com</td>
			<td>59-169</td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila Stock bottles, 6 oz square bottom w/ Flugs</td>
			<td>geneseesci.com</td>
			<td>32-130BF</td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila vials, Narrow K-Resin super bulk</td>
			<td>geneseesci.com</td>
			<td>32-118SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry active yeast</td>
			<td>Genesee Scientific</td>
			<td>62-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>IBI Scientific</td>
			<td>IB15720</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ Basic Anesthesia System</td>
			<td>World Precision Instruments</td>
			<td>EZ-175</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Centrifuge tubes</td>
			<td>Corning</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon round bottom tubes</td>
			<td>Corning</td>
			<td>352057</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine point Sharpie marker</td>
			<td>Sharpie</td>
			<td>30001</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Nutating Mixer</td>
			<td>Fisher Scientific</td>
			<td>88-861-043</td>
			<td></td>
		</tr>
		<tr>
			<td>Flugs-Narrow Plastic Vials</td>
			<td>Genesee Scientific</td>
			<td>49-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Thermometer</td>
			<td>Cole-Palmer</td>
			<td>EW-08008-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen hose</td>
			<td>Thermo Scientific</td>
			<td>398202</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen tank-Dewar</td>
			<td>Cooper Surgical Inc</td>
			<td>900109-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen transfer vessel</td>
			<td>Electron Mircoscopy Sciences</td>
			<td>61891-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Paintbrushes(Red Sable) Size #0</td>
			<td>Electro Microscopy Sciences</td>
			<td>66100-00</td>
			<td>This is used to separate the flies via sex without causing injury.</td>
		</tr>
		<tr>
			<td>Plastic funnel</td>
			<td>Plews and Edelmann</td>
			<td>570-75-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Polarizing light microscope</td>
			<td>Microscope Central</td>
			<td>1100100402241</td>
			<td>Used to more clearly view Drosophila during sexing</td>
		</tr>
		<tr>
			<td>ProPette Pipette Tips</td>
			<td>MTC Bio Incorporated</td>
			<td>P5200-100U</td>
			<td></td>
		</tr>
		<tr>
			<td>ProPette Pipette Tips</td>
			<td>MTC Bio Incorporated</td>
			<td>P5200-1M</td>
			<td></td>
		</tr>
		<tr>
			<td>ProPette Pipette Tips</td>
			<td>MTC Bio Incorporated</td>
			<td>P5200-5M</td>
			<td></td>
		</tr>
		<tr>
			<td>Propionic Acid</td>
			<td>Sigma Aldrich</td>
			<td>P1386-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Rayon Balls</td>
			<td>Genesee Scientific</td>
			<td>51-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Reynolds wrap standard aluminum foil</td>
			<td>Staples</td>
			<td>1381273</td>
			<td></td>
		</tr>
		<tr>
			<td>Roaster Oven (Crockpot)</td>
			<td>Hamilton Beach</td>
			<td>32950</td>
			<td></td>
		</tr>
		<tr>
			<td>Scotch 810 Magic Tape</td>
			<td>Electron Microscopy Sciences</td>
			<td>77300</td>
			<td></td>
		</tr>
		<tr>
			<td>Spray bottle with trigger</td>
			<td>US Plastic</td>
			<td>66446</td>
			<td>Used to spray ethanol to clean work bend areas</td>
		</tr>
		<tr>
			<td>Tegosept</td>
			<td>Genesee Scientific</td>
			<td>20-258</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Drosophila Incubator</td>
			<td>Thermo Scientific</td>
			<td>3990FL</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Revco 4 degree Lab fridge</td>
			<td>ThermoFisher Scientific</td>
			<td>REL7504D</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Revco Lab Freezer</td>
			<td>ThermoFisher Scientific</td>
			<td>REL7504A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Anatrace</td>
			<td>T1003-1-GA</td>
			<td></td>
		</tr>
	</tbody>
</table>

circadian entrainment of drosophila melanogaster

Nearly universal among organisms, circadian rhythms coordinate biological activity to earth&#39;s orbit around the sun. To identify factors creating this rhythm and to understand the resulting outputs, entrainment of model organisms to defined circadian time-points is required. Here we detail a procedure to entrain many Drosophila to a defined circadian rhythm. Furthermore, we detail post-entrainment steps to prepare samples for immunofluorescence, nucleic acid, or protein extraction-based analysis.

Controlled circadian entrainment allows researchers to examine biology at specific time points throughout the circadian day using the ZT1-ZT19 timing schedules or to add time-points as necessary. Here we use light and darkness to entrain flies to circadian cycles and verify entrainment by immunoblotting and immunofluorescence analysis of the period protein, a marker for circadian entrainment (Figure 1). Upon correct entrainment, period proteins should have a characteristic intensity and mobility pattern (Figure 1A) and should be visible at specific locations in the ZT1 brain (Figure 1B). Although other variables, including food and temperature, can influence circadian entrainment, light is most simple and reliable to control17. For the purpose of these methods, incubator temperatures are kept constant, relying on cerebral clock neurons that are influenced by light for entrainment18.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61176/61176fig1.jpg" /> 
Figure 1: Verification of entrainment. (A) Immunoblotting of whole cell extracts prepared from heads of entrained flies shows canonical patterns of period protein mobility and intensity19. 1.4 female heads from each of the indicated Zeitgeber times (ZT) were analyzed using an anti-Per antibody. (B) Immunofluorescence of entrained brains collected at ZT, where the Period protein is found in a characteristic pattern (bottom panels, recreated from Helfrich-Forster20). Shown are images taken from different sections of the brain, capturing all neurons expected to contain Period protein at ZT1. Scale bar is 40 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61176/61176fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ZT1</td>
			<td>ZT7</td>
			<td>ZT13</td>
			<td>ZT19</td>
		</tr>
		<tr>
			<td>Light between 10 am - 10 pm</td>
			<td>Light between 10 am - 10 pm</td>
			<td>Dark between 9 am - 9 pm</td>
			<td>Dark between 9 am - 9 pm</td>
		</tr>
		<tr>
			<td>Collected at 11 am after 1 hour in light</td>
			<td>Collected at 5 pm after 7 hours in light</td>
			<td>Collected at 10 am after 1 hour in dark</td>
			<td>Collected at 4 pm after 7 hours in dark</td>
		</tr>
	</tbody>
</table>
Table 1: ZT1-ZT19 Circadian Rhythm Timing Schedules.

Watch this Scientific Journal Video about Circadian Entrainment of Drosophila Melanogaster at JoVE.com

Circadian Entrainment of Drosophila Melanogaster

Here, we detail how to synchronize Drosophila to a circadian day. This is the first, and most important step necessary for studying biological rhythms and chronobiology.

Nearly universal among organisms, circadian rhythms coordinate biological activity to earth&#39;s orbit around the sun. To identify factors creating this ...

circadian-entrainment-of-drosophila-melanogaster

Research

JoVE Journal

Biology

4.2K Views.  University of Missouri - Kansas City. Here, we detail how to synchronize Drosophila to a circadian day. This is the first, and most important step necessary for studying biological rhythms and chronobiology.

Video: Circadian Entrainment of Drosophila Melanogaster

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Dissection z larw CNS na Drosophila melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

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 ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...