Measuring Plasma Cytokine Levels in Postnatal Mice

Measurement of the levels of cytokines within plasma collected at post-natal day 7 and 120 was performed using Luminex 200 system (Luminex, Austin, Tex.). Plasma concentrations of interleukin (IL) -2, interferon-γ (IFN-γ), IL-4, IL-5, IL-13, and IL-17A were assessed using the Milliplex Assay (Millipore) system. The study method was modified from previously reported methods [24] (link), [25] (link). Antibody conjugated beads were incubated first with diluted standards or plasma from animal subjects for 2 hours and then with detector antibodies for 1 hour at room temperature. Fluorescent detection was performed after the sample had been incubated for 1.5 hours with biotin as reporter and incubated for 30 min with fluorescent dye-conjugated streptavidin-phycoerythrin. Cytokine levels were measured by using a flow cytometer and were analyzed with Flowmetrix software (Master Plex QT 1.2 system) [24] (link).