For lifespan analysis, flies were maintained in vials at a density of 25 flies per vial on standard cornmeal/molasses medium.
CRP standard was reconstituted by adding 1 ml of the 1 x diluent buffer into the vial of lyophilized standard and vortexed vigorously.
For these treatments, samples of about 30 flies were placed in glass vials containing 7 ml of standard medium, and the vials were submerged in water baths at the temperatures described.
The flies were reared in a chamber, which allowed simultaneous culturing of more than 20,000 culture vials of flies under standard conditions (25°, approximately 60% relative humidity and 16-hr light:8-hr dark).
The concentrations of these compounds in gaseous headspace were found to substantially decrease in comparison to previously developed hydrocarbon pump oil based vials; hence, the amount of standard loaded onto SPME fibers was at most, half that of the previous vial design.
Larvae were raised on fly food supplemented with all-trans retinal (Sigma, 100-200 nmol added daily on top of vials containing ∼5 ml of standard medium) until the third instar stage, then dissected in HL3 buffer [30].
We reared flies in vials containing 10 ml of standard cornmeal, agar, sugar, and yeast medium at a constant temperature of 25°C, 60 75% relative humidity, and a 12-hr light-dark cycle.
Pupae were picked and placed in isolation in individual 16×100 mm glass vials containing 1.5 ml of standard food medium, where they were allowed to emerge as adults and kept for 5 7 days before testing.
Larvae were raised to the third instar stage on fly food supplemented with all-trans retinal (Sigma, 100 200 nmol added daily on top of vials containing ∼5 ml of standard medium) before testing their response to short (1 2 second) illumination periods using blue light at an intensity of about 0.2 mW/mm2.
