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On the following day, the vials were transferred to liquid nitrogen and stored there for a duration of 2 days up to 4 weeks before use.
After different periods of anoxia, vials were transferred back to the air lock and then to room atmosphere.
After 10 minutes, all flies had fallen into stupor and the vials were transferred to the main chamber which was equilibrated with pure N2.
The vials were transferred to a programmable freezer (Planner Products, Cryobiosystem) and cooled at 2°C/min for seeding and followed by 0.3°C/min to −40°C and subsequently at 10°C/min to −140°C.
Vials were transferred to -80°C for storage pending analysis.
Vials were transferred to room temperature and the time to recovery from coma recorded.
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At day 11.25, a randomized sample of 1792 adults from these vials are transferred, in groups of 16 pairs, to 56 'adult competition vials' with a prescribed amount (3.84 mg) of live yeast on top of the culture medium.
The suspension of the needed number of vials was transferred with a cannula (18G) into two 50 ml disposable sterile syringes (Perfusion Plastipak, Becton Dickinson, Heidelberg, Germany).
The contents of one iTRAQ reagent vial were transferred to one sample tube.
The 8 flies in the vial were transferred to a new vial with fresh food every other day.
The eight flies in the vial were transferred to a new vial with fresh food every other day for a total of 30 days.
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