Lipidomic Analysis of Drosophila Tissues

Four batches of 5 flies each were mixed with 700 µl MTBE/methanol (10/3, v/v) in 2 ml safe-seal micro tubes (Cat#: 72.695.500, Sarstedt, Nürmbrecht, DE) and disrupted with a metal bead (5 mm diameter; Cat#: 504942, Askubal Korntal-Münchingen, DE) in a Retsch MM 400 mixer mill (3 min, 30 Hz, 4°C) and lipids were extracted by shaking for 20 min at 1400 rpm and 4°C in a ThermoMixer (Eppendorf, Hamburg, DE). After the addition of 200 µl H₂O, samples were again incubated 20 min at 1400 rpm and 4°C. Phase separation was induced by centrifugation for 10 min at 16,000 x g and 4°C. The upper organic phase was collected and dried under a stream of nitrogen. Lipids were washed by dissolving in 500 µl chloroform/methanol (2/1, v/v) and again dried under a stream of nitrogen and stored at −20°C or prepared immediately for LC-MS analysis. For MS analysis, lipid extracts were dissolved in 0.5 ml chloroform/methanol (2/1, v/v), 50 µl of the sample were diluted with 100 µl isopropanol and 10 µl were injected for analysis using UPLC-QTOF-MS as described (Knittelfelder et al., 2014 (link)). Data analysis was performed using the Lipid Data Analyzer (LDA) software (Hartler et al., 2011 (link)). The abundance of each TG species was normalized to the intensity of the internal standard TG 51:0 (Larodan, Solna, SWE) and to animal number.