Peritoneal Macrophage Isolation and Culture

Peritoneal macrophages from C57BL/6 mice were prepared according to the method described by Zhang et al. (49 (link)), with some modifications. In brief, mice were injected with 1 mL of a sterile solution of thioglycollate-enriched medium. After 4 days, the animals were euthanized and immediately injected with 5 mL sterile ice-cold 5 mM EDTA in PBS into the peritoneal cavity, and the peritoneal fluid was collected. Cells were centrifuged at 1,400 rpm twice for 5 min, and the pellet was resuspended in RPMI medium. For most experiments, peritoneal macrophages were seeded at 1 × 10⁶ cells/mL in 6-well plates and incubated with hemocyanin (1 mg/mL each) for 24 h. Then, the supernatants were collected to determine the cytokine levels. The same controls as listed above were used. iBMMs from C57BL/6 WT and TLR4 KO mice, which were generated using J2 transforming retroviruses (expressing Raf and Myc), as described previously (50 (link)), were a gift from Prof. D. Golenbock (University of Massachusetts Medical Center, USA). iBMMs were seeded at 1.5 × 10⁵ cells/mL in 96-well plates and harvested after 24 h of incubation with 1 mg/mL FLH. The supernatants were assessed for cytokine levels. LPS from Escherichia coli serotype R515 (Enzo Life Sciences) was used as a positive control, and PBS was used as a negative control.