The TAG assay was adapted from [72 (link)]. Multiple batches of 10 male flies each were processed for TAG analysis. A small scoop of zirconium beads and 300 μl of PBS, 0.05% Tween was added to each tube. The samples were then homogenised using a Precellys homogeniser. For protein estimation, 40 μl of homogenised sample was immediately collected and frozen. The remaining sample was heat inactivated for 10 minutes at 70°C. A volume of 200 μl of the heat-inactivated sample was transferred to fresh tubes. A volume of 4 μl of lipase (25 KU/mL; Merck; 437707) was added to each tube and mixed. The samples were then kept at 37°C overnight. After overnight incubation, the samples were centrifuged at 14,000 rpm for 3 minutes, and the supernatant collected. Each sample was then put in triplicates in a 96-well plate. Different concentrations of glycerol were used as standards. Prewarmed Free Glycerol reagent (Merck; F6428) was then added to the 96-well plate, and the plate was incubated at 37°C for 6 minutes. After a brief spin, absorbance measurements were taken at 540 nm using a Hidex Sense Plate Reader. The Bradford assay was used for protein estimation. Briefly, pre-heat-inactivated sample was centrifuged, and the supernatant put in triplicates in a 96-well plate. Bradford reagent was then added, and absorbance measurements taken at 600 nm.
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