Triglyceride solution

Total body triacylglycerols (TAGs) in flies were measured using the coupled colorimetric assay as described previously42 (link)43 (link). Glyceryl trioleate served as the TAG standard. Five females (or eight males) per group were weighed and homogenised in 1 ml of 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, Waltham, MA, USA) at 37 °C for 30 min with shaking. Absorbance was read at 562 nm, and the quantity was estimated using the standard curve. Each measurement was performed with at least three biological replicates.
Glycogen was determined using the Glycogen Assay Kit (Sigma, Deisenhofen, Germany), according to the manufacturer’s instructions. Homogenisation was performed with five animals per sample using the Bead Ruptor 24 (BioLab Products). Six biological replicates were analysed for each condition, and mean values ±SEM are given.

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.

The whole fly bodies were collected and fixed in 4% paraformaldehyde for 30 min at room temperature. After washing with phosphate-buffered saline, the flies were repeatedly frozen in liquid nitrogen and thawed on ice three times, followed by staining with a solution containing 1 μg/ml BODIPY dye (Invitrogen, Darmstadt, Germany) for 1 h in the darkness before observation by epifluorescence microscopy (Olympus, Hamburg, Germany).
Total body triacylglycerols (TAGs) in flies were determined using a coupled colorimetric assay method as described previously (Hildebrandt et al., 2011 (link); Hoffmann et al., 2013 (link); Li et al., 2016 (link)). Briefly, eight males (or five females) per group were weighed and homogenized in 1 ml 0.05% Tween-20 using a Bead Ruptor 24 (BioLab Products, Bebensee, Germany). Homogenates were heat-inactivated for 5 min at 70°C and incubated with triglyceride solution (Fisher Scientific, Waltham, MA, USA) at 37°C for 30 min with mild shaking. The absorbance was read at 562 nm and glyceryl trioleate served as a TAG standard.