A Milliplex MAP Mouse Cytokine Magnetic Bead Panel multiplex assay (Millipore Sigma) was used to detect 26 cytokines, chemokines, and growth factors in mouse tissue homogenates from vaccinated and control mice at 4 days post footpad MAYVBeAr challenge. Cytokines from tissues of AdV-MAYV and AdV-GFP vaccinated mice were analyzed using a R&D Systems mouse magnetic Luminex LXSANSN-26 assay. Briefly, 25 μl of clarified tissue homogenate from ipsilateral and contralateral ankles and quadriceps (N = 10 per group), calf (N = 8 per group), and naïve mouse calf and ankles (N = 4 per tissue). The manufacturer’s protocol was followed with minor alterations as previously described [39]. The plate was read on a Luminex 200 Detection system (Luminex).

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