Mouse Cytokine Multiplex Assay

A Milliplex MAP Mouse Cytokine Magnetic Bead Panel multiplex assay (Millipore Sigma) was used to quantify the protein levels for 26 cytokines, chemokines, and growth factors in ankle tissues from vaccinated and control mice at 8 days post footpad challenge with CHIKV. Entire ankle samples were homogenized in PBS with 2 mm beads (Propper Manufacturing Co., Inc.) using a Precellys 24 homogenizer (Precellys Bertin Technologies). Cellular debris was pelleted by centrifugation at 5,000 × g for 2 min, and the supernatants were transferred to a new 1.7 ml tube. For the assay, washed antibody-conjugated polystyrene magnetic beads were incubated with a seven-point standard curve or 25 μl of ankle tissue homogenate plus 25 μl of blocking buffer. Samples were incubated at room temperature in the dark for 2 h, washed and then labeled with biotinylated detector antibody for 1 h. Following washing with blocking buffer, the beads were incubated with Phycoerythrin-conjugated streptavidin for 30 min and then washed. Cytokines were quantified using a Luminex 200™ Detection system (Luminex), and the data was analyzed and graphed using GraphPad Prism v6 software.