G1152

L1 worms were grown on wars-1 RNAi or the control RNAi (HT115). To treat worms with a high concentration of Trp, the L4 stage worms grown on HT115 bacteria were transferred to NGM plates supplemented with 100 mM L-tryptophan (ab146400, Abcam). After 24 h of exposure, adult worms were collected from the plates and were washed 3 times with M9. Worms were then lysed using 200 mg of 180 µm glass beads (G1152, Sigma-Aldrich) and a bead-beating apparatus (BEAD MILL 4, Fisher Scientific) in 250 µl of water. Bead-beating was done six times, with each round lasting 30 s at maximum speed (5 m/s), with 20-second intervals on ice. To extract the metabolites, 700 µl of acetonitrile (34851, Honeywell) was added to each tube to have the final acetonitrile concentration of 70%. The bead-beating process was repeated six times, each lasting 30 s at maximum speed (5 m/s). Samples were then transferred to fresh 2 ml tubes and centrifuged at 21,000 g for 10 min at 10 degrees Celsius to pellet protein. The clear supernatants, which contain metabolites, were isolated. The protein pellets were used to measure the protein content using the Bradford assay, which was later used to normalize the metabolomics data.

Pellets with 5 × 10⁶ uninfected or infected macrophages were re-suspended in 350 µL of cold methanol/water (3:1, v:v) and 25 mg of glass beads (710–1180 µM, G1152, SigmaAldrich, Germany), followed by four cycles of frost/defrosting in a liquid N₂/37 °C bath. The cells were disrupted at 50 mHz for 10 min in TissueLyser LT (Qiagen, Germany). The samples were clarified by 15,700× g for 10 min at 4 °C centrifugation and the supernatant was collected and evaporated to dryness by SpeedVac SPD121P (Thermo Fisher Scientific, Waltham, MA.) at 35 °C for 2 h. After this, the solid residue was re-suspended in 60 µL 0.1 M formic acid with 0.2 mM of methionine sulfone, homogenized for 15 min in a vortex, and centrifuged at 15,700× g for 15 min at 4 °C. The 35 µL of supernatant was transferred to polypropylene vials (Agilent Techno Vials, Waldbronn, Germany) for analysis. Quality-control (QC) samples were prepared by pooling equal volumes of all the samples and they were analyzed along the analytical sequence, to evaluate the stability and performance of the instruments during measurements [27 (link)].