D5 lxa4

PCM prepared in phenol red-free media was added to 100% methanol (1:2) and stored at −80°C. Similarly, phenol red-free media from RAW264.7 cells exposed to PCM +/− PGE₂ antibody for 6 hours was added to 100% methanol and stored at −80°C. These samples were then subjected to solid-phase extraction followed by targeted LC-MS/MS analysis as previously described in detail [16 ]. Extraction recovery was aided by deuterium-labeled internal standards for each chromatographic region including d4-PGE₂ and d5-LXA₄ (Cayman Chemical), and lipid mediators were identified and quantified using multiple reaction monitoring transitions and full MS/MS spectra as compared with authentic external standards (Supplemental Figure 3). For visualization, a heatmap of all identified lipid mediators was constructed using Metaboanalyst (www.Metaboanalyst.ca). For this, a missing value imputation was first performed followed by a generalized log transformation and autoscaling. Finally, Ward clustering algorithm and Euclidean distance measures was applied to data. The resulting heatmap illustrates the relative change in abundance of individual mediators across each experimental condition.

Samples were taken to solid-phase extraction (SPE) using Isolute C18 SPE 3 mL cartridges (Biotage, USA), as in Colas et al.^{15 (link)}. Briefly, internal standards (d8-5-HETE, d5-RvD2, d5-LXA₄, d4-LTB₄, d4-PGE₂; 500 pg each; Cayman Chemicals, USA) were added along with four volumes of methanol before SPE, and covered on ice for 30-60 minutes to allow for protein precipitation. During SPE, 6 mL of water was eluted through each cartridge, followed by elution of 6 mL of hexane. Lipid mediators were collected by elution and collection of 6 mL of methyl formate. Methyl formate fractions from SPE were analyzed by a liquid chromatography-tandem mass spectrometry system, QTrap 5500 (AB Sciex) equipped with a Shimadzu LC-20AD HPLC (Tokyo, Japan). A Poroshell 120 EC-18 column (100 mm × 4.6 mm × 2.7 μm; Agilent Technologies, USA) was kept in a column oven maintained at 50°C, and lipid mediators (LMs) were eluted in a gradient of methanol/water/acetic acid from 55:45:0.01 (v/v/v) to 98:2:0.01 at 0.5 mL/min flow rate. In order to monitor and quantify the levels of targeted LMs, multiple reaction monitoring (MRM) was used with MS/MS matching signature ion fragments for each molecule (at least six diagnostic ions; ~0.1 pg limits of detection) and standard curves (r²>0.98 for each lipid mediator and pathway marker).