2 tedc

Fura-2 acetoxymethyl ester; 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF); bis-(1,3-dibutylbarbituric acid) trimethine oxonol (oxonol), 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA; reactive oxygen and nitrogen species (ROS/RNS) sensor) and 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1), were purchased from Molecular Probes (Life Technologies, USA). Arachidonic, oleic, docosahexanoic, palmitic acid, indomethacin (inhibitor of cyclooxygenase, Indomet), nordihydroguaiaretic acid (inhibitor of lipoxygenase, NDGA), valinomycin, luciferin, luciferase, rhodamine 123, salts and buffers were obtained from Sigma-Aldrich (St. Louis, MO, USA). 2-(1-thienyl)ethyl 3,4-dihydroxybenzylidenecyanoacetate (inhibitor of lipoxygenase, 2-TEDC), was purchased from Tocris Bioscience (through Gene-Xpress, Chile).
indomethacin, NDGA and 2-TEDC have been shown to be cell permeant and have 50% inhibitory concentrations of 0.1–0.2 μM for indomethacin [26 (link)], 0.5–1.0 μM for NDGA [27 (link)] and 0.09, 0.013 and 0.5 μM (5-, 12-, and 15-lipoxygenases, respectively) for 2-TEDC [28 (link)].

Dulbecco’s Modified Eagle Medium/Ham’a F12 (DMEM/F12 1:1) and bovine calf serum were purchased from Life Technologies Corporation (Carlsbad, CA, USA). Ketoconazole, Indomethacin, SQ 22536, H-89, lipopolysaccharides from Escherichia coli 055:B5 and forskolin were purchased from Sigma-Aldrich (St. Louis, MO, USA), and normal goat serum was acquired from Dako (Carpentaria, CA, USA). Anti-Iba-1 antibody and anti-glial fibrillary acidic protein (GFAP) antibody were purchased from Wako (Richmond, VA, USA) and Sigma-Aldrich (St. Louise, MO, USA), respectively. Antibodies to PKA, CREB, and GAPDH were purchased from Cell Signaling. Heneicosanoic acid was from Nu-Chek Prep (Elysian, MN). Butylated hydroxytoluene (BHT), LY311727, neomycin sulfate, and 10% boron trifluoride-methanol (BF₃/methanol) were from Sigma-Aldrich (St. Louis, MO). Methyl arachidonyl fluorophosphonate (MAFP), bromoenol lactone (BEL), and ZLJ-6 were obtained from Cayman Chemical (Ann Arbor, MI). 2-TEDC was from Tocris Bioscience (Bristol, UK). HPLC grade water and acetonitrile were from Avantor Performance Materials (Center Valley, PA). Other solvents, BCA protein assay kit, and acetic acid were from Fisher Scientific (Pittsburgh, PA)

Unless specified below, all chemical reagents were of research grade and were obtained from Merck (Kenilworth, NJ, USA). Those chemicals sourced from Thermo Fisher Scientific (Waltham, MA, USA) included BODIPY 581/591 C11 and the LIVE/DEAD viability reagent. The lipoxygenase inhibitors, PD146176, BW-B-70C and 2-TEDC were purchased from Tocris Bioscience (Avonmouth, Bristol, UK). ML355 and docosapentanoic acid were supplied by Cayman Chemical (Ann Arbor, MI, USA). Anti-ALOX15 antibodies were purchased from Abcam (Cambridge, MA, USA), while GE Healthcare (Chicago, IL, USA) supplied nitrocellulose membranes and Percoll. Tris-HCl was obtained from ICN Biochemicals (Castle Hill, NSW, Australia) and Hams-F10 was bought from MP Biomedicals (Irvine, CA, USA). The catalogue numbers of antibodies and details for the working solutions of each inhibitor and probe are provided in Supplementary Table S1.