Thermo tracefinder software

At day 16 of culture, 3 × 1.10⁶ cells of each culture condition were separated from stimulation beads, washed twice in PBS and stored at −80 °C. The extraction solution was composed of 50% methanol, 30% acetonitrile, and 20% water. After the addition of extraction solution, the samples were vortexed for 5 min at 4 °C and then centrifuged at 16,000 × g for 15 min at 4 °C. The supernatants were collected and stored at −80 °C until analyses. Liquid chromatography–mass spectrometry analyses were conducted on a QExactive Plus Orbitrap mass spectrometer equipped with an Ion Max source and a HESI II probe and coupled to a Dionex UltiMate 3000 UPLC system (Thermo Fisher Scientific) as previously described^{66 (link)}. Data were acquired using Thermo Xcalibur software (Thermo Fisher Scientific). The peak areas of metabolites were determined using Thermo TraceFinder software (Thermo Fisher Scientific), identified by the exact mass of each singly charged ion, and by known retention time on the HPLC column.

After stimulation by CinA, metabolites extraction was performed on 7.5 × 10⁵ cells with a solution of 50% methanol, 30% acetonitrile, and 20% water. After the addition of extraction solution (1 mL per 2 × 10⁶ cells), samples were vortexed for 5 min at 4 °C and then centrifuged at 16,000× g for 15 min at 4 °C. The supernatants were collected and stored at −80 °C until analysis. Liquid chromatography–mass spectrometry analyses were conducted on a QExactive Plus Orbitrap mass spectrometer equipped with an Ion Max source and a HESI II probe and coupled to a Dionex UltiMate 3000 UPLC system (Thermo Fisher Scientific). The metabolites were detected across a mass range of 75–1000 m/z at a resolution of 35,000 (at 200 m/z), and data were acquired with Thermo Xcalibur software version 4.3 (Thermo Fisher Scientific). The peak areas of the metabolites were determined using Thermo TraceFinder software (Thermo Fisher Scientific), identified by the exact mass of each singly charged ion and by the known retention time on the high performance liquid chromatography (HPLC) column.

The peak areas of different metabolites were determined using Thermo TraceFinder software (Thermo Scientific, Waltham, MA, USA) where metabolites were identified by the exact mass of the singly charged ion and by known retention time on the HPLC column. Commercial standards of all metabolites detected had been analyzed previously on this LC-MS system with the pHILIC column. The ¹³C-labeling patterns were determined by measuring peak areas for the accurate mass of each isotopologue of many metabolites. Intracellular metabolites were normalized to protein content of the cells, measured at the end of the experiment by the Lowry assay.