Cytokine Profiling in Toxoplasma-Infected Murine Cells

C57BL/6 BMDMs were differentiated and stimulated as previously described (77 (link)). Briefly, the cells were seeded (2 × 10⁵ per well) in 96-well plates and left to adhere overnight at 37°C in 5% CO₂. Cells were infected with freshly lysed GRA83^3xHA, ∆gra83, and GRA83c tachyzoites at an MOI of 0.5, and supernatants (200 µL) were collected at 18 h after infection and stored at −80°C. For in vivo assays, C57BL/6 mice were euthanized after 48 h of infection, and the peritoneal cavity was washed with 1 mL of PBS, spun at 400 × g for 10 min to pellet the cells. The supernatant was collected and stored at −80°C. Spleens were also collected at 48 h, mechanically disrupted by a tissue homogenizer (Ika) in 1 mL of PBS with protease inhibitor cocktail (Roche), and spun at 10,000 × g for 10 min to pellet debris. Supernatants were collected and stored at −80°C. IL-12p40 and IFN-γ levels were determined using commercially available enzyme-linked immunosorbent assay kits (BD Biosciences) according to the manufacturer’s instructions.