9 10 dihome

For naïve T cell and DC co-cultures, naive CD4⁺ T cells (5 × 10⁵ cells/mL) and DCs (5 × 10⁵ cells/mL) were co-cultured in 96-well U-bottom plates and stimulated with soluble anti-CD3ε mAb (5 μg/mL) supplemented with 0.1 ng/mL TGF-β1 and 10 ng/mL IL-2 (BioLegend) for 3 days. For naïve T cell monocultures, naive CD4⁺ T cells (5 × 10⁵ cells/mL) were cultured in 96-well flat-bottom high-binding plates (Corning). The cells were stimulated with immobilized anti-CD3ε mAb (5 μg/mL) and soluble anti-CD28 mAb (5 μg/mL) supplemented with 0.1 ng/mL TGF-β1 and 10 ng/mL IL-2 (BioLegend) for 3 days. LA, 10,13(OH)18:0, 17,18-DiHETE, 9,10-EpOME, 9,10-DiHOME, and 9,10-EpOME were purchased from Cayman Chemical Company and stored at -30 °C in methyl acetate before use. For all experiments, the methyl acetate was removed under a nitrogen steam. The compound was resuspended in TexMACS medium (Miltenyi Biotec) containing 20 µM GlutaMAX (Thermo Fisher Scientific), 55 µM 2-mercaptoethanol, 10 mM HEPES, 100 U/mL penicillin, and 100 µg/mL streptomycin, or RPMI 1640 medium containing 10% FBS, 20 µM GlutaMAX (Thermo Fisher Scientific), 55 µM 2-mercaptoethanol (Thermo Fisher Scientific), 10 mM HEPES, 100 U/mL penicillin, and 100 µg/mL streptomycin. The methyl formate fraction dissolved in ethanol was diluted in the culture medium at the same ethanol concentration (0.5%) under all dilution conditions.

Vasicine, vasicinone, isoorientin, isovitexin, vitexin, apigenin, 12,13-DiHOME, 9,10-DiHOME, 13(S)-HODE, 9(S)-HODE, 13-oxoODE, 9-oxoODE, 12,13-EpOME and 9,10-EpOME, 9-HpODE, and 7-ethoxyresorufin were purchased from Cayman Chemical (Ann Arbor, MI, USA). AITC, ionomycin calcium salt, HC-030039, and A967079 were from Sigma-Aldrich (St. Louis, MO, USA). DMSO was from Fisher Scientific (Waltham, MA, USA).