Cell Culture

Macrophages were obtained as follows: 1 ml thioglycollate (4%) was intraperitoneally injected into mice and mice were sacrificed at the fourth day post injection. The belly skin was opened by pulling the skin apart at the middle of abdomen and abdominal membrane was not broken during the procedure. A 10 ml syringe attached to an 18G needle was used to inject 10 ml RPMI-1640 (with 10% FBS) into the abdominal cavity. The media were then slowly withdrawn back into the syringe and this was subsequently injected into 6 well plate (1.5–2 ml per well) and incubated at 37°C for 2 h. Supernatant was aspirated and then 2 to 3 ml fresh media was added before being incubated overnight. On the next day, the cells were harvested. Macrophages isolated from PAR-2^-/- and Control mice were cultured in RPMI-1640 supplemented with 100 μg/ml of penicillin, 100 μg/ml of streptomycin and 10% FBS. The cells were incubated in an atmosphere of 5% CO₂ at 37°C and were routinely passaged every 3 days. To investigate the effects of PAR-2 on macrophage activation, we assessed the gene expression of TNF-α, IFN-γ, IL-6 and MCP-1 in unstimulated and LPS-stimulated macrophages that express or lack PAR-2.