Macrophage preparation and characterization
Twenty female BALB/c mice were obtained at six to eight weeks of age from the Pasteur Institute of Iran, and maintained in the animal laboratory in accordance with the Ethical Commission of Tarbiat Modares University guidelines. For preparation of peritoneal macrophages, 3 ml of 4% w/v thioglycollate medium was injected into the peritoneal cavity of the BALB/c mice. After four days, the macrophages were harvested by injecting; and thereafter, harvesting the fresh cold DMEM from the peritoneal cavity near the fat region of the lower abdominal area took place. Harvested peritoneal fluid was centrifuged for five minutes at 350×g and the resultant cell pellets were seeded (1×106 cells/ ml) to 4 or 24 well plates for downstream analysis and incubated in a humidified incubator (37˚C and 5% CO2). The medium of newly isolated macrophages was changed after six hours for separating the non-adhesive cells. Phenotypic analysis of isolated cells was performed by flow cytometry for CD14+ and CD11b+ markers (Figure S2, Supporting Information ) as described previously [26].