Thermo scientific triglycerides reagent

All metabolic analyses were performed as previously described^{73 (link),74 (link)}. For TAG assay, 25 staged larvae or six adult flies were collected and homogenized in 1xPBS containing 0.1% Tween 20 and TAG was quantified using Thermo Scientific™ Triglycerides Reagent (Thermo Fisher Scientific, Waltham, MA, USA). For glycogen measurement, samples were digested with amyloglucosidase (Sigma-Aldrich, St Louis, MO, USA) and glucose contents were quantified using Thermo Scientific™ Glucose Hexokinase Reagents (Thermo Fisher Scientific, Waltham, MA, USA). The relative level of each metabolite was obtained by normalizing the metabolites to total protein.

Lipids were extracted from 50-200 mg of tissue by O/N incubation at 55°C in ethanolic KOH (66.6% 96% ethanol with 33.3% KOH). Samples were brought to a volume of 1,200 μL with 50% ethanol then centrifuged (5 min, maximum speed) in a microcentrifuge. After mixing 100 μL of supernatant with 100 μL of 0.5 M MgCl₂, samples were kept on ice for 10 min. Samples were then centrifuged (5 min, maximum speed) in a microcentrifuge and supernatant was transferred to a new tube. Glycerol standards were prepared ranging from 1,000 mg/dL down to 1 mg/dL. In a standard 96-well plate, 3 μL of sample or standard was mixed with 300 μL of Thermo Scientific™ Triglycerides Reagent (Thermo Fisher Scientific, TR22421) and incubated at 37°C for 10 min before reading on CLARIOstar Plus plate reader (BMG Labtech). Triolein equivalents (TE) were fitted using a least squares (ordinary) fit line calculated with GraphPad Prism software. TE measurements were normalized to tissue weight.