Cayman 17 18 epete

Contact hypersensitivity was induced as described previously.10 Briefly, the abdominal skin of mice was shaved, after which 25 μL of 0.5% (vol/vol) 1‐fluoro‐2,4‐dinitrofluorobenzene (DNFB, Nacalai Tesque) dissolved in a mixture of acetone (Nacalai Tesque) and olive oil (Nacalai Tesque) at a ratio of 4:1 was applied. After 5 days, both sides of the right and left ears were challenged with 0.2% (vol/vol) DNFB (10 μL at each site). After another 2 days, ear thickness was measured using a micrometer (model MDC‐25MJ 293‐230, Mitsutoyo). In order to evaluate the effects of 17,18‐EpETE, mice received racemic compound of 17(S),18(R)‐EpETE and 17(R),18(S)‐EpETE ((±)17,18‐EpETE), a commercially available Cayman (±)17,18‐EpETE (Cayman Chemical). In some experiments, mice were treated with stereoselective 17(S),18(R)‐EpETE (>99% enantiomeric excess) or 17(R),18(S)‐EpETE (>99% enantiomeric excess), which were purified from synthesized (±)17,18‐EpETE, or BM‐3 17(S),18(R)‐EpETE. These lipids were injected intraperitoneally into mice by 100 ng/animal at 30 minutes before DNFB treatment. In some experiments, BM‐3 17(S),18(R)‐EpETE were injected intraperitoneally into mice by 1 µg, 100 ng, or 10 ng/animal in order to evaluate dose response. We used 0.5% (vol/vol) ethanol dissolved in PBS (Nacalai Tesque) as a vehicle control.

Neutrophils were purified from bone marrow as described previously.10 Briefly, bone marrow‐derived neutrophils were harvested using 62% Percoll. For the actin polymerization assay, purified neutrophils (4 × 10⁵ cells) were suspended in HBSS (Nacalai Tesque) containing 0.2% bovine serum albumin (Sigma Aldrich) and allowed to adhere to fibronectin‐coated coverslips (Neuvitro) for 15 minutes at 37°C in a 5% CO₂ incubator. Neutrophils were treated with either 1000, 100, 10, or 1 nmol/L of commercially available Cayman (±)17,18‐EpETE, BM‐3 17(S),18(R)‐EpETE, 18‐HEPE (Cayman Chemical), RvE1 (Cayman Chemical), or 0.03% (vol/vol) ethanol (vehicle control) for 15 minutes and then stimulated with 1 μmol/L N‐formyl‐methionyl‐phenylalanine (fMLP; Sigma Aldrich) or 100 nmol/L leukotriene B₄ (LTB₄; Cayman Chemical) for 2 minutes at 37°C in a 5% CO₂ incubator. Neutrophils were fixed in 4% paraformaldehyde (Nacalai Tesque), permeabilized using 0.5% (vol/vol) Triton X‐100 (Nacalai Tesque) in PBS, and stained with 100 nmol/L Acti‐stain 488–phalloidin (Cytoskeleton) for 30 minutes at room temperature. Finally, cell nuclei were stained by incubating neutrophils with 4ʹ,6‐diamidino‐2‐phenylindole for 30 seconds at room temperature. Images were obtained with Leica TCS SP8 confocal microscopy (Leica Microsystems).