Macrophage-Spleen Cell Coculture Assay

Coculture of Mφ with naive spleen cells was performed as follows. Adherent Mφ among peritoneal exudate cells (PECs) from healthy or 8 weeks after STZ-treatment Wt or Mif−/− mice were obtained. Briefly, the Mφ density was adjusted to 5 × 10⁶ cells/mL, and the cells were plated (100 μL) in 96-well flat-bottom plates (Costar, Cambridge, MA, USA). Three hours later, the PECs were washed three times with warm sterile PBS to remove nonadherent cells, and 10 μg of OVA (Worthington, USA) in 100 μL of DMEM supplemented media was added. Three hours later, adherent Mφ were washed three times with warm sterile DMEM to remove excess OVA that had not been phagocytosed. Spleen cells from OVA-transgenic mice were obtained as previously described [45 (link)], suspended at 1 × 10⁶ cells/mL, and added (100 μL) to PECs at a ratio of 1 Mφ : 5 spleen cells. The cocultures were maintained at 37°C in 5% CO₂ for 5 days. Then, [3H]-thymidine (185 GBb/mmol activity, Amersham, UK) was added at 0.5 μCi/well, and the cells were incubated for a further 18 h. The cells were harvested using a 96-well harvester (Tomtec, Toku, Finland) and then counted using a microplate counter (Trilux, Toku, Finland). The values are presented as counts per minute (CPM) from triplicate wells.