Multiplex Cytokine Measurement Protocol

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-γ), IFN-γ -inducible protein (IP)-10, interleukin (IL)-1RA, IL-6, and tumor necrosis factor (TNF)-α were measured using a human cytokine / chemokine magnetic bead panel (Milliplex MAP kit, Merck Millipore, Billerica, MA, USA), a Magpix Luminex instrument and Xponent software (version 4.2 Luminex Corp, Austin, Texas, USA) according to manufacturer's instructions. Standard curves using a 5-parameter logistic regression were applied to calculate concentrations of cytokines. Antigen- and mitogen-induced cytokine production was calculated by subtraction of non-stimulated background concentrations from sample concentrations. Measurements below the limit of quantification were set to 0.1 pg/ml, measurements above the limit of quantification were set to 10,000 pg/ml (calibration range: 3.2–10,000 pg/ml). A valid positive control was defined as an uncorrected cytokine concentration > 20 pg/ml (26 (link)). A valid negative control was defined as uncorrected cytokine concentration < 20 pg/ml. In cases where the nil concentration was higher than 20 pg/ml, the positive control had to be higher than the nil concentration.