Metabolite Measurement in Drosophila Larvae

Measurements of total body glucose and lactate were performed on a pool of 5 larvae per genetic condition using a fluorescence-based enzymatic detection kit (Biovision Inc.) as described before [81 (link)]. Briefly, larvae were collected in 1.5 ml microcentrifuge tubes, the excess of water was removed and samples were frozen stored at -80°C until further analysis. Upon defrosting, 15 μl PBS per larvae was added and tissues were disrupted with a sterile pestle (Axygen), on ice. Samples were spun at 14,000 x g for 10 min, at 4°C. The supernatant was immediately used for metabolite measurements according to manufacturers’ protocol. Fluorescence readings (Ex/Em 535/590) were taken with the FLUOstar Omega instrument (BMG Labtech). Results were read from a linear regression curve based on the dilutions of the standard. Statistical analysis between indicated groups was performed using the unpaired t-test and considered significant at P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***).