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.