Synchronizing Drosophila to define circadian times is the first step in studying events that either control or respond to the biological clock. Synchronization of all the models will follow the same basic approach. There are many steps to do this practical and it may seem overwhelming to put them all together.
Expect to try a few times before it feels natural. Keep temperatures stable and I want the light out during the three minutes steps. Prepare the fly food in a Crock-Pot at 121 degrees Celsius, mixing as ingredients are added.
Cover the Crock-Pot and bring the contents to a rolling boil, mixing the contents every 10 minutes. After 20 minutes at a rolling boil, turn off the heat, and add 83.6 milliliters of water to the Crock-Pot. Leave the lid off as the food cools.
While the food is cooling, stir it every 10 minutes to avoid allowing a film to form on the top. Also measure the temperature every 10 minutes with a glass thermometer. Once the food has cooled to below 60 degrees Celsius, add Tegosept and propionic acid, as described in the manuscript.
Mix well and adjust the Crock-Pot to maintain a temperature, just a few degrees less than 60 degrees Celsius. Use a peristaltic pump to distribute the fly food. For narrow vials, pump 10 milliliters, and for six ounce square bottom bottles, pump 60 milliliters.
Store wild type flies at 25 degrees Celsius for 10 to 12 days, until approximately 200 pupae are attached to the insides of the bottle. To remove the adults from the bottle, tip them into a new bottle, and use the dull end of a number zero paintbrush to push any remaining adults in. Wipe the paintbrush with 70%ethanol before and after use.
Incubate the pupae at 25 degrees Celsius to allow the next generation to emerge. After three days, the flies will be zero to three days old. Remember that temperature, light, and even your choice of food will influence entrainment.
Be sure to keep them, each control during your experiment. At the designated time points for collection, use a carbon dioxide anesthesia pad to immobilize the flies, and collect 100 males and 100 females. Female flies are much larger than males.
The flies can also be differentiated by examining genitalia. Males have dark rounded genitalia, whereas females have lighter, more pointed genitalia. Place males and females in separate vials with 100 females per vial and 50 males per vial.
Male social interactions lead to many deaths when there are 100 individuals in a vial. To enable circadian entrainment to occur, place the vials in light regulated incubators for three to five days. To produce flies for future entrainment, place 25 of the remaining females, and five to seven of the remaining males in a new bottle.
Incubate the bottle at 25 degrees Celsius. For every 100 flies that will be collected. Prepare a narrow vial by adding 4.8 milliliters of fixative.
Place the narrow vials in a bucket of ice, and take the bucket, paper towels, foil, and tape to the incubator. To collect the flies, place a funnel in the narrow vial, then remove the cap from the vial of flies and quickly invert it into the funnel. Tap gently to help guide the flies into the vial of fixative.
When collecting males, combine two vials of 50 flies into one narrow vial. Wrap the vials for ZT13 and ZT19 in foil. After transferring the flies, tape the caps in place to avoid spillage.
Place the vials on the Nutating Mixer at 165 RPM, and four degrees Celsius, for four hours. The flies are no longer light sensitive, so, the foil may be removed to verify that the flies are being submerged in the fixative. After removing the vials from the Nutating Mixer, remove the formaldehyde, then wash the flies three times with 3000 microliters of 1X PBS, inverting the vial during each wash.
Store the vials at four degrees Celsius, to await future immunofluorescence. Light and darkness were used to entrain flies to circadian cycles. Entrainment was verified by immunoblotting, and immunofluorescence analysis of the period protein, a marker for circadian entrainment.
Immunoblotting of whole cell extracts, prepared from heads of entrained flies shows a canonical pattern of period protein mobility and intensity. Immunofluorescence of entrained brains, collected at ZT1, shows period proteins in characteristic pattern. This method is a jumping off point for immunofluorescent, immunoplotting, immunoprecipitation, behavioral analysis, and any other technique you may want to use to compare how pathway changes as the biological clock ticks.
With the ability to entrain your model organism, you can ask if your favorite pathway changes over time, and whether the effects in your favorite pathway can lead to disruption of the biological. A number of diseases are worsened when the biological clock is disrupted. Identifying the underlying mechanisms will help us create more effective medicines for people suffering from diseases, such as Alzheimer's and Parkinson's disease.
