Multiplex analysis of cytokines and chemokines
Concentrations of TNF-α, IL-1β, IL-6, IL-18, and INF-γ were simultaneously measured in 25 μL of medium from homogenized cells using a Milliplex M.A.P. kit Mice Cytokine/Chemokine Magnetic Bead Panel (Millipore, Billerica, MA) by following manufacturer’s instructions. Antibody immobilized beads were detected on a Luminex 100 xMAP technology machine (Austin, TX). Standard curves were generated for each cytokine/chemokine using standards included in the kit for serum samples. The median fluorescence intensity for each analyte was calculated using a five-point logistic parameter curve, and normalized to the amount of protein in each sample.