Cytokine measurements by ELISA

For in vitro cytokine determination, BMDMs were seeded overnight at a density of 2 × 10⁵ cells/well in 48-well plates and prestimulated with 300 ng/ml of PAM(3)CSK(4) (Invivogen) for 4 h, and subsequently infected with MAYV. The cytokines in the supernatants were assayed using a mouse IL-1β ELISA kit (BD Biosciences) according to the manufacturer’s instructions. For in vivo cytokine determination, footpads were processed and the supernatants from total homogenates were obtained. The levels of IL-1β and IL-18 in the homogenates were detected using a mouse IL-1β ELISA kit (BD Biosciences) and a mouse IL-18 ELISA kit (RD-MBL), respectively, according to the manufacturer’s instructions. Detection of IL-1β, IL-18 and Caspase 1 (p20 subunit) in human serum samples was accomplished by using human IL-1β ELISA kit II (BD Biosciences), human total IL-18 (RD) and human caspase-1 ELISA kit (RD) respectively.