Colorimetric Quantification of Fly Triacylglycerols

Total body triacylglycerols (TAGs) in flies were measured using the coupled colorimetric assay (CCA) method as described previously [19 (link),21 (link)]. Glyceryl trioleate (T7140, Sigma-Aldrich, Taufkirchen, Germany) served as the TAG standard. Five females (or 8 males) per group were weighted and homogenized in 1 mL 0.05% Tween-20 using a Bead Ruptor 24 (BioLab Products, Bebensee, Germany). Homogenates were heat-inactivated at 70 °C for 5 min, centrifuged, and incubated with triglyceride solution (Fisher Scientific, Schwerte, Germany) at 37 °C for 30 min with shaking. The absorbance was read at 562 nm and the quantity estimated using the standard curve. Each measurement was performed with at least three biological replicates.