Quantifying Triacylglycerol Levels in Flies

The level of triacylglycerol (TAG) was measured as described previously (Tennessen et al., 2014 (link)). Briefly, 5 female adult flies per vial were weighed and frozen at −80°C until assay. After homogenizing in PBS + 1% Triton-X, the samples were heated at 70°C for 10 min and then cooled to room temperature. An aliquot was added to the triglyceride reagent (T2449; Sigma, United States), incubated at 37°C for 60 min, added to the free glycerol reagent (F6428; Sigma, United States), and incubated for 5 min at 37°C. TAG content was quantified using a microplate spectrophotometer at OD540 using a standard curve and blank well.