4.6.1. Sample Preparation for Lipidomics Analysis

Briefly, cells from four wells of 6-well plates were harvested after experiments and transferred into 1.0 mL of ice-cold 80% methanol in water (*v*/*v*) to perform homogenization using the TissueLyser II homogenizer (Qiagen) at the frequency of 30/sec, in 6 × 30 s bursts, and 20 s in between to avoid high temperature. An aliquot (20 μL) of cell homogenates was saved separately for future protein content measurement by BCA (bicinchoninic acid) assay (Thermo Scientific, Rockford, IL, USA). The remaining homogenate from each cell sample was added to 10 μL of IS mixture stock solution (5 μg/mL for AA-d8, 2 μg/mL for DHA-d5 and EPA-d5, and 0.5 μg/mL), then agitated on ice in the dark for 1–2 h. For cell culture media, 1 mL of culture media sample was mixed with 1.5 mL of ice-cold methanol and 10 μL of IS mixture stock solution, then agitated on ice in dark for 15 min. After incubating the homogenate or culture media sample on ice, samples were centrifuged at 6000× *g* at 4 ◦C for 10 min to remove any precipitated proteins. All LM standards and isotope-labelled LM internal standards were purchased from Cayman Chemical Co (Ann Arbor, MI, USA). Formic acid (reagent grade, ≥95%) was obtained from Sigma-Aldrich. HPLC-MS grade acetonitrile, water, methanol, and ethanol were purchased from J.T. Baker (Phillipsburg, NY, USA).

Both cell and culture media samples were cleaned and concentrated by Solid Phase Extraction (SPE) before being injected into the LCMS. Before loading samples to the preconditioned SPE cartridges (Strata-X 33 μm polymeric reversed phase, Phenomenex, Torrance, CA, USA), 4 or 6 mL of ice-cold 0.1% formic acid was added in the supernatant from cell or medium sample, respectively, to fully protonate the LM species. Once the sample had been totally loaded, cartridges were washed with 1 mL of 0.1% formic acid followed by 1 mL of 15% (*v*/*v*) ethanol in water to remove excess salts. Then the LMs from the SPE sorbent bed were eluted by methanol. Solvents were removed using an Eppendorf® 5301 concentrator centrifugal evaporator (Eppendorf, Hauppauge, NY, USA), and the dried extracts stored at −80 ◦C for future LC-MS/MS analysis.

#### 4.6.2. LC-MS/MS Conditions

All components of LC-MS/MS system are from Shimadzu Scientific Instruments, Inc. (Columbia, MD, USA). LC system was equipped with four pumps (Pump A/B: LC-30AD, Pump C/D: LC-20AD XR), a SIL-30AC autosampler (AS), and a CTO-30A column oven containing a 2-channel six-port switching valve. The LC separation was conducted on a C8 column (Ultra C8, 150 × 2.1 mm, 3 μm, RESTEK, Manchaca, TX, USA) along with a Halo guard column (Optimize Technologies, Oregon City, OR, USA). The MS/MS analysis was performed on Shimadzu LCMS-8050 triple quadrupole mass spectrometer. The instrument was operated and optimized under both positive and negative electrospray and multiple reaction monitoring modes (+/− ESI MRM). The settings of flow rate and gradient program for the LC system as well as MS/MS conditions are recommended by a software method package for 158 lipid mediators (Shimadzu Scientific Instruments, Inc., Columbia, MD, USA) and further optimized following our previously published quantification method [57]. Briefly, the optimized conditions are as follows: Interface voltage, 4.0 kV; interface temperature, 275 ◦C; DL temperature, 275 ◦C; heating block temperature, 400 ◦C; drying gas (N2), 10 L/min; nebulizing gas (N2), 3 L/min; heating gas (Air), and 10 L/min; CID gas (Ar), 230 kPa. The acquisition was divided into multiple segments. The *m*/*z* transitions and their tuning voltages were selected based on the best MRM responses from the instrumental method optimization software. All analyses and data processing were completed on Shimadzu LabSolutions V5.91 software (Shimadzu Scientific Instruments, Inc., Columbia, MD, USA).
