We started by placing our vial full of adult flies in the refrigerator in a bucket of ice for at least 15 minutes, until the flies stopped moving, then moved this vial to our ice bucket to prepare to sort the flies. At the same time we labeled 4 plastic vials with our group name, lab section, and the date and treatment (appropriate ecdysone hormone concentrations), and put 2 grams of dehydrated fly media in the bottom of each tube, then added 10 mL of water or the appropriate concentration of reagent (control (water), 0.1, 1.0, and 10 µM of ecdysone), and mixed to create blue, uniform medias in each vial. Then we added flies to the vials by using a soft brush to gently sweep two male and two female flies into each separate vial, inserting cotton stoppers, and laying the vials on their sides until the flies regain consciousness. The vials were then placed into incubators.
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