Lipopolysaccharide lps from escherichia coli serotype 0111 b4

For the extract characterization, grade HPLC acetonitrile and methanol were purchased from Merck (Germany). For the behavioral experiments, the following drugs were used: imipramine from Henrifarma (Porto Alegre, Brazil) and lipopolysaccharide (LPS) from Escherichia coli serotype 0111:B4 from Sigma Chemical Co. (St. Louis, MO, USA). All chemicals were obtained in the highest grade.

The RPMI‐1640 or DMEM medium and fetal bovine serum (FBS) were purchased from Gibco, Inc. (Carlsbad, CA, USA). Phorbol 12‐myristate 13‐acetate (PMA) and lipopolysaccharide (LPS) from Escherichia coli (serotype 0111:B4) were obtained from Sigma‐Aldrich (St. Louis, MO, USA). Goat antimouse SDC1 antibody, and human tumour necrosis factor alpha (TNF‐α, Cat. DTA00C), interleukin‐1beta (IL‐1β, Cat. DLB50), cytokine‐induced neutrophil chemoattractant ELISA kits (CINC‐1, CXCL‐1, Cat. QC101) were all purchased from R&D Systems, Inc. (Minneapolis, MN, USA). NF‐κB P65 (8242) and NF‐κB p65 (3033) antibody were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Mouse anti‐human SDC1 antibody and CD15 antibody was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX ,USA) and ZSGB‐Bio Inc. (Beijing, China) respectively. Human Sdc1 ELISA kit was purchased from Elabscience Biotechnology Co., Ltd (Cat. E‐EL‐H1298; Wuhan, China).

Macrophage differentiation was performed according to previously established protocols [23] (link). THP-1 cells (5×10⁶) were seeded in 75-cm² tissue culture flasks and cultured with 320 nM phorbol 12-myristate 13-acetate (PMA; Sigma) for 4 h. For M2-type macrophage differentiation, THP-1 cells were treated with 320 nM PMA, 20 ng/mL interleukin (IL)-4, and 20 ng/mL IL-13 (PeproTech, Rocky Hill, NJ, USA) for another 20 h. To generate M1-type macrophages, THP-1 cells were treated with 320 nM PMA for 4 h, and then treated with 320 nM PMA, 20 ng/mL interferon (IFN)-γ (PeproTech), and 100 ng/mL lipopolysaccharide (LPS) from Escherichia coli serotype 0111:B4 (Sigma) for 20 h. After stimulation, cells were washed with phosphate-buffered saline (PBS) twice and cultured with fresh medium for 24 h, and then CM was collected.
PC-3 prostate cancer cells were seeded at 5×10⁵ in 6-well plates 1 day prior to treatment. To treat cells, cells were placed in medium containing different concentrations of EPA, DHA (0, 25, and 50 µM) or 10 µM GW9662 for 24 h, and then cells were treated with CM containing the same concentration of fatty acids for another 24 h. PC-3 cells were harvested for further analysis.