1290 infinity 2 multisampler

Aortas were harvested, weighed, snap-frozen in liquid nitrogen, and then kept at −80°C. Frozen aortas were homogenized with dry ice–chilled 80% methanol followed by centrifugation at 10,000g for 5 minutes at 4°C. The supernatant was collected and an aliquot of a volume equivalent to 10 mg of the tissue from each sample was saved at −80°C. All aliquots were dried via speed vac for mass spectrometry (MS) processing. The Agilent 1290 UHPLC system and Agilent Technologies Triple Quad 6470 Mass Spectrometer (LC-MS/MS) with a 1290 Infinity II LC Flexible Pump (Quaternary Pump), 1290 Infinity II Multisampler, and 1290 Infinity II Multicolumn Thermostat with a 6-port valve were used for metabolomics analysis. Data were collected using parameters published previously (93 (link)–95 (link)). Agilent MassHunter Workstation Software LC/MS Data Acquisition for 6400 Series Triple Quadrupole MS with version B.08.02 was used for compound optimization and sample data acquisition. Agilent MassHunter Workstation Quantitative Analysis for QQQ version 10.1, build 10.1.733.0, was used to integrate and quantitate ion abundance peak areas. Absolute itaconate concentrations were calculated using an itaconate (93598, Sigma-Aldrich) standard curve.

Freshly isolated primary B cells (10⁶ cells/well) were cultured with previously γ-irradiated hCD40L-3T3 cells in the presence or absence of NS1 (1 μg/mL) for 16 h at 37°C. After incubation, cells were collected by centrifugation at 500 × g for 5 min at 4°C. Cell pellets were resuspended in ice-cold 80% methanol and kept at −80°C for 10 min. The supernatants were then collected after centrifugation at the highest speed for 5 min at 4°C. Metabolites were dried at 4°C using a SpeedVac. Metabolite pellets were reconstituted in 50 μL of 50% methanol, and 40 μL was transferred to an autosampler glass vial for untargeted LC-MS analysis. Samples were run on an Agilent 1290 Infinity II LC-6470 triple-quadrupole (QqQ) tandem mass spectrometry (MS/MS) system with the following parameters. The Agilent Technologies triple-quadrupole 6470 LC-MS/MS system consists of the 1290 Infinity II LC flexible pump (quaternary pump), the 1290 Infinity II multisampler, the 1290 Infinity II multicolumn thermostat with a 6-port valve, and the 6470 triple-quadrupole mass spectrometer. Agilent MassHunter Workstation software LC-MS data acquisition for 6400 series triple-quadrupole MS with version B.08.02 was used for compound optimization, calibration, and data acquisition.