Triglycerides and glycogen quantifications

       Eight 5-days old males (with heads removed) were collected, frozen in liquid nitrogen and stored at -80°C. Flies were ground in 200µl of PBST buffer (PBS with 0.01% Triton X-100). 30µl of extract were transferred to another vial for protein measurement. The rest of the extract was heated at 70°C for 10 minutes (Tennessen et al., 2014). 

       For TAG measurement, 6µl of heat-treated homogenate were mixed with 25µl of PBS and 30µl of TAG reagent (Pointe Scientific, Cat. T7531) or Free Glycerol Reagent (MilliporeSigma, Cat. F6428). Triglyceride standard solution (from Pointe Scientific, Cat. T7531 kit) and glycerol standard solution (MilliporeSigma, Cat. G7793) were used as standards. Reactions were incubated for 30 minutes at 37°C, centrifuged 6000g for 2 minutes and supernatants were transferred to 96-well plate, after which absorbance was read at 540nm. The TAG concentration in each sample was determined by subtracting the values of free glycerol in the corresponding sample. 

       For glycogen measurement, heat-treated homogenate was centrifuged 5 minutes at 10000g. 6µl of supernatant were mixed with 24µl of PBS and 100µl of glucose reagent (MilliporeSigma, Cat. GAGO20) with or without the addition of amyloglucosidase (MilliporeSigma, Cat. A1602, 0.25U per reaction) and transferred to 96-well plate. Glycogen solution (Fisher Scientific, Cat. BP676-5) and glucose solution (MilliporeSigma, Cat. 49161) were used as standards. Reactions were incubated 60 minutes at 37°C, after which 100µl of sulfuric acid were added to stop the reaction, and the absorbance was read at 540nm. Glycogen concentration in each sample was determined by subtracting the values of free glucose in corresponding sample. 

       To measure total protein, 30µl of extract (before heat-treatment) were centrifuged for 5 minutes at 6000g. 6µl of supernatant were added to 800µl of water and 200µl of Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, Cat. 5000006). Vials were incubated for 5 minutes and absorbance were read at 595nm. BSA was used as a standard.