Lipid Extraction and Quantification in Drosophila

Fly lipids were extracted according to Bligh and Dyer [36] (link). Five flies per replicate were homogenized in 150 µl methanol, 75 µl chloroform and 60 µl water in a Bioruptor sonifier (15 min with alternating 45 sec on/off intervals, low intensity setting; www.diagenode.com) or in the peqlab Precellys 24 instrument (10 sec 5000 rpm) using 1.4 mm ceramic beads (peqlab 91-PCS-CK14S). Lipids were extracted from the homogenates for 1 hour at 37°C before 75 µl chloroform and 75 µl 1 M KCl were added. Phase separation was achieved by centrifugation (Eppendorf 5417C; 2 min 3000 rpm) and the chloroform phase solvent was evaporated in a SpeedVac concentrator (Thermo Savant ISS110). Lipid pellets were resuspended in 60–70 µl chloroform/methanol (1∶1). For fat extraction after CCA the samples were extracted with 500 µl methanol and 250 µl chloroform for 15 min at 37°C before adding 250 µl chloroform and 250 µl 1 M KCl and lipid recovery as described above. Lipid extracts from CCA samples were separated by TLC as described below using 20 µg each of triolein, pentadecanoin and stearic acid as lipid standards.
Lipids extracted from 1 mg fly wet weight were separated on high performance thin layer chromatography (HPTLC) plates (Merck 105633) using n-hexane/diethylether/acetic acid (70∶30∶1, v/v/v; Merck) as liquid phase along with the following standard lipids: triolein (TAG; Sigma T7140), pentadecanoin (DAG; Sigma D8508), stearic acid (FA; Fluka 85679). Plates were air dried, dipped into 8% (w/v) H₃PO₄ containing 10% (w/v) copper (II) sulfate pentahydrate and charred for 10 min at 180°C on a hot plate (Gerhard H22 electronic). Fly lipid classes were quantified by photodensitometry (FujiFilm LAS-1000 and Image Gauge V3.45) scaled to a dilution series of the corresponding lipid standard (5–80 µg triolein; 1–16 µg pentadecanoin).
Depicted in Fig. 3A are representative experiments with average values of triplicate measurements and corresponding standard deviations. Experiments were repeated at least twice. Shown in Fig. 3B are average values of triplicate measurements of two independent experiments.
To determine the glyceride composition of fly homogenates the TAG and DAG content of flies was determined by TLC and the free glycerol content by CCA. Relative abundance of the glyceride classes was calculated using the following (average) molecular weights: glycerol (92,1 g/mol), triglycerides (844,96 g/mol), diglycerides (562,5 g/mol) and expressed as nmol/ mg fly wet weight.