Lipid Mediator Characterization Protocol

Synthetic LXA₄ was purchased from EMD Millipore (Billerica, MA) and RvD1 was purchased from Cayman Chemical, Ann Arbor, MI). Both compounds were dissolved in ethanol as supplied by the manufacturer and were stored at −80 °C with minimal exposure to light. Immediately prior to use, each SPM was diluted in with Krebs-Ringer bicarbonate buffer with HEPES (KRB-HEPES, 119 mM NaCl, 4.8 mM KCl, 1.0 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, 10 mM HEPES, and 5.5 mM glucose [pH 7.45]) to the desired concentrations and added to the cells. The LXA₄ analogs were prepared as described previously ^{16 (link), 17 (link)}. The cells were incubated at 37 °C in the dark. Daily working stock dilutions were discarded following each experiment. Annexin A1 (AnxA1) was purchased from MyBiosource (San Diego, CA). Aristolochic acid, 2-aminoethyl diphenylborinate (2-APB), 1 butanol and t-butanol were from Sigma Aldrich (St Louis, MO) while U0126 was purchased from R&D Systems (Minneapolis, MN). H89 and R0-31317549 were obtained from EMD Millipore (Billerica, MA).