Formula 5 24 instant drosophila medium

A culture of D. melanogaster Canton S line was used for targeted proteomics analysis. The flies were kindly provided by Dr. Natalia Romanova (Moscow State University, Department of Biology). The line was kept on Formula 5-24 instant Drosophila medium (Carolina Biological Supply Company, USA) in 50 mL disposable plastic test tubes.
For targeted proteomic analysis, brains of third stage larvae (L3), late pupae (P12-14 according to [66] (link)) and young adult flies (approximately 7 days old) of both sexes were used. The culture was maintained in the dark at 25°C. Brain dissection for all phases was performed as described previously [16] (link). Briefly, alive insects were taken out, and the body was rapidly removed by a needle. The head was placed into 0.01 M PBS at pH 7.4 (Sigma-Aldrich, USA), and the head capsule was torn apart by two forceps under visual control through a stereo microscope (Nikon SMZ645, Japan) with 10 × 1 magnification. The extracted brains were collected into the same buffer solution, and then centrifuged at 6000 g for 15 min (Centrifuge 5415R, Eppendorf, Germany). PBS was removed, and the brain pellet was frozen at -80 °C until sample preparation.
Two biological replicates were obtained independently with a time gap of six months.

Live samples of Drosophila melanogaster Canton S line were kindly provided by Dr. Natalia Romanova from Moscow State University, Department of Biology. The flies were kept on Formula 5-24 instant Drosophila medium (Carolina Biological Supply Company, USA) in 50 ml disposable plastic test tubes. The initial temperature for the fly culture was 25 o C. The flies had been transferred to a new tube as they reached the adult stage. For general samples that were used for shotgun proteome analysis, as well as for the initial targeted analysis. Two hundred flies were selected from the culture regardless of their age. For the second MRM experiment 80 male flies were used, 72 hours old.