Effect of curcuminoids on lipid mediator profiles of activated M1 and M2 polarized macrophages

Human macrophages of the M1 and M2 phenotype were pre-treated with 3b or 2f for 15 min and then stimulated with Staphylococcus aureus-conditioned medium for 180 min, and lipid mediator profiles were determined by UPLC-MS/MS. 
Raw data including the absolute values (ng) and normalized data (% of control) of all individual experiments was uploaded.
The methods and results were published in Rao et al., Biochem Pharmacol. 2022 Sep:203:115202. doi: 10.1016/j.bcp.2022.115202 
Human M1 or M2 macrophages were treated with vehicle (0.1 % DMSO), compound 3b or 2f in PBS pH 7.4 plus CaCl2 (1 mM) for 15 min and then stimulated with Staphylococcus aureus-conditioned medium (SACM, 1 %) for 180 min at 37 °C. SACM was prepared. In brief, Staphylococcus aureus (strain 6850) was grown for 18 h with orbital shaking (150 rpm) at 37 °C in brain heart infusion medium (Carl Roth, Karlsruhe, Germany). The conditioned medium was harvested after centrifugation (3350 × g, 10 min) and sterile filtered using a Millex-GP Syringe Filter Unit (0.22 μm; Merck).
Cell supernatants (1 mL) were collected and mixed with ice-cold methanol (2 mL). d8-5S-HETE, d4-LTB4, d5-LXA4, d5-RvD2, d4-PGE2 (200 nM, 10 µL each) and d8-AA (10 µM, 10 µL) were added as internal standards. Lipid mediators were extracted by solid phase extraction using Sep-Pak C18 6 cc Vac Cartridges (500 mg; Waters). Briefly, samples were stored at −20 °C for at least 45 min to allow protein precipitation. After centrifugation (1200 × g, 4 °C, 10 min), the supernatant was combined with acidified water (pH 3.5, 7 mL) and loaded onto pre-equilibrated solid phase cartridge columns. Washing steps with water and n-hexane (6 mL, each) followed. Lipid mediators were eluted with methyl formate (6 mL), brought to dryness using an evaporation system (TurboVap LV, Biotage, Uppsala, Sweden) and resuspended in methanol/water (50/50, v/v, 100 µL) for UPLC-MS/MS analysis.
For metabololipidomics analysis, lipid mediators were separated at 50 °C on an Acquity UPLC BEH C18 column (130 Å, 1.7 µm, 2.1 mm × 100 mm, Waters). The Acquity Ultraperformance LC system (Waters) was operated at a flow rate of 0.3 mL/min using a mobile phase consisting of methanol, water, and acetic acid (42:58:0.01, v/v/v), which was ramped to 86:14:0.01 (v/v/v) over 12.5 min followed by isocratic elution at 98:2:0.01 (v/v/v) for 3 min. Eluted lipid mediators were detected by (scheduled) multiple reaction monitoring using a QTRAP 5500 mass spectrometer (Sciex, Framingham, MA), which was equipped with an electrospray ionization source that was operated in negative mode. Acquired mass spectra were processed using Analyst 1.6.2 (Sciex).