Evaluating IFNγ-Induced Antigen Presentation

To prepare the target cells, 50 units/mL mouse IFNγ (Prospec Protein Specialists) was added to the B16-OVA cell culture. Forty-eight hours later, the cells were fixed with 1% paraformaldehyde and washed before being added to the coculture. For immune cell stimulation, splenocytes (5 × 10⁵ cells/well) were incubated with prefixed target cells (B16-OVA, 5 × 10⁴ cells/well) or one of the following peptides at 2 μg/mL: mgp100 (refs. 24–32 (link); AnaSpec), OVA (257–264; Invitrogen), TRP-2 (180–188; AnaSpec), or GFP (118–126; Biomatik). Twenty-four hours after incubation in a round-bottom 96-well plate, the concentration of IFNγ in the medium was assessed with a mouse IFNγ DuoSet ELISA kit (R&D Systems).