Cell lysis and extraction of lipid mediators was performed as previously described (Schultz et al., 2020 (link)). In brief, internal standards were added and the cells were lysed using FastPrepTM lysing matrix D and a FastPrepTM homogenizer. Lysing cycle was repeated after a washing step. Combined supernatants were used for an alkaline hydrolysis followed by a solid-phase extraction.
Samples were measured with an Agilent® HPLC system (1200), coupled to an Agilent® 6460 Triple quadrupole mass spectrometer with a Jetstream ionization source using a dynamic multiple reaction monitoring (dMRM) method. Chromatographic and MS parameters are referred to in (Schultz et al., 2020 (link)). Data analysis was performed with Agilent Mass Hunter Qualitative and Quantitative Analysis software (version B.08.00 for both). For all detected oxylipins, a relative quantification was done by normalizing the area of the metabolite signal to the area of the signal of the internal standard compound (relative amount). 12-HETE-d8 was the internal standard for HETEs, EETs, HEPEs and HDHAs, and 13-HODE-d4 for HODEs, and 13-HOTrE. All relative amounts of the oxylipins were normalized to the cell number (106 cells per sample).
Samples were measured with an Agilent® HPLC system (1200), coupled to an Agilent® 6460 Triple quadrupole mass spectrometer with a Jetstream ionization source using a dynamic multiple reaction monitoring (dMRM) method. Chromatographic and MS parameters are referred to in (Schultz et al., 2020 (link)). Data analysis was performed with Agilent Mass Hunter Qualitative and Quantitative Analysis software (version B.08.00 for both). For all detected oxylipins, a relative quantification was done by normalizing the area of the metabolite signal to the area of the signal of the internal standard compound (relative amount). 12-HETE-d8 was the internal standard for HETEs, EETs, HEPEs and HDHAs, and 13-HODE-d4 for HODEs, and 13-HOTrE. All relative amounts of the oxylipins were normalized to the cell number (106 cells per sample).