Bone marrow derived dendritic cells (BMDC) were generated from RIG-I+/+ and RIG-I-/- mice and T cell re-stimulation experiments were performed as previously described[31 (link)], [32 (link)]. Briefly, BMDC were first infected with PR8 at an MOI of 0.5 for 5h, washed with PBS 3 times to remove free virus, and resuspended in Iscove's Modified Dulbecco's Media complete media with FBS (IMDM, Invitrogen). For some experiments, infected BMDC were treated with IFN-I (200U/ml) before T cells were added. T cells from naïve or IAV infected RIG-I+/+ and RIG-I-/- mice (day 7 post infection) were enriched using Pan T cells isolation kit II (Miltenyi Biotec) and co-cultured with PR8 infected BMDC at a ratio of 10:1 for 2–3 hours followed by the addition of Brefeldin A (5μg/ml; eBiosciences). DC-T cell co-cultures were further incubated for an additional 8-10hrs. The cells were first surface stained for cell surface markers, followed by intracellular staining for cytokines. For intracellular staining, cells were incubated in permeabilization and fixation buffer (BD Pharmingen) for 45 minutes followed by 2 washes in 1xPBS, 1%FBS, 0.5% Saponin (Sigma, St Louis, MO) and intracellular staining with anti-IFNγ (2μg/ml, XMG1.2) (Biolegend), anti- Granzyme B (2μg/ml, GB11) (Biolegend), anti-TNFα (2μg/ml, MP6-XT22) (eBiosciences) for 30 minutes.
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