2.6. Assay for Cytokine Release from Peritoneal Macrophages

Macrophage activity was measured by slightly modifying the method described by Suzuki et al. [28]. After intraperitoneal injection of 2 mL of 5% thioglycollate medium into BALB/c mice (male, 6–8 weeks old, Coretech) and recovery of the induced macrophages within 72–96 h, the cells were washed 2–3 times with RPMI 1640. The cell number was adjusted to 1 × 10⁶ cells/mL, of which 100 μL was dispensed into a 96-well plate. The cells were then cultured in a 5% CO₂ incubator (37 °C, 2 h) to form a macrophage monolayer, the supernatant was removed, and the non-adherent macrophages were washed with Rosewell Park Memorial Institute (RPMI) 1640-fetal bovine serum (FBS) medium. Then, the adherent phagocytes were incubated for 24 h with the indicated concentrations of EPS. The respective concentrations of interleukin (IL)-6, IL-12, and tumor necrosis factor (TNF)-α in the culture supernatants were measured with enzyme-linked immunosorbent assay (ELISA) kits (Pharmingen, San Jose, CA, USA).