Glycerol standard

For triacylglyceride (TAG) content quantification, two whole female flies were homogenized in 0.05% Tween20 according to [61 (link)]. 10 replicates were used per condition. TAG content was quantified using the Triglyceride Infinity Reagent (ThermoScientific) using Glycerol standards (Sigma). Protein content was determined using the BCA protein assay reagent (Pierce). Student’s t test (Excel) was used to assess statistical difference between two conditions, while one-way ANOVA (GraphPad) was used for > 2 conditions.

Five flies were collected for each biological sample, and 6–8 replicates were prepared for all control and experimental groups. The flies were homogenized in 100 μL of cold PBST (pH = 7.2, 0.05% of Tween 20). After 10 min incubation at 70 °C, 20 μL of the homogenized sample was separately added to either 20 μL of PBST, for measuring the free glycerol, or 20 μL of triglyceride reagent (Sigma-Aldrich, Stockholm, Sweden), for measuring the total glycerol content. All samples were then incubated at 37 °C for 60 min and then centrifuged for 3 min at 14,000 rpm. After incubation, 30 μL of each individual sample was transferred to a 96-well plate and mixed with 100 μL of free glycerol reagent (Sigma-Aldrich, Stockholm, Sweden). The plate was incubated for 5 min at 37 °C. The absorbance of each sample was measured with a Multiscan GO spectrophotometer (Thermo Scientific, Stockholm, Sweden) at 540 nm and calculated according to the standard curve. The standard curve was obtained from a serial dilution of glycerol standards (Sigma-Aldrich, Stockholm, Sweden) and was produced along with the samples. The TAG concentration was determined as TAG concentration = total glycerol concentration − free glycerol concentration.