Milliplex cytokine kits

Naïve murine T helper cells were negatively selected from spleen, isolated from IL-21R^-/- or WT C57Bl6/J mice using a mouse naïve CD4⁺ T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Naïve CD4⁺ T cells were cultured for four days in 6-wells plates at 37°C, 5% CO₂, in RPMI-1640 medium containing 5% fetal calf serum (FCS), 50 μm β-mercaptoethanol, 50 μg/ml gentamycin, and 1% pyruvate. The following Th17 stimulation cocktail was added, with or without recombinant mouse (rm)IL-6 (50 ng/ml; eBioscience, San Diego, CA, USA): anti-CD3 (5 μg/ml; eBioscience; adsorbed to the wells over night at 4°C), anti-CD28 (2.5 μg/ml; eBioscience), anti-IL-2 (5 μg/ml; eBioscience), TGF-β (1 ng/ml; eBioscience), IL-1β (10 ng/ml; kind gift of Pfizer), and TNFα (10 ng/ml; eBioscience). Differentiation efficacy was determined with flow cytometry. Supernatant cytokine levels were determined using the Luminex multianalyte technology, in combination with Milliplex cytokine kits (Merck Millipore, Darmstadt, Germany).

A total of six cytokines, including interferon (IFN)-γ, interleukin (IL)-13, IL-1β, IL-6, tumor necrosis factor (TNF)-α, and IL-12 (p40) were tested using Milliplex Cytokine Kits (Merck Millipore, Burlington, MA, USA). The fluorescence of cytokines was measured using a Luminex 200 system and analyzed by Milliplex Analyst software (version 5.1.0.0) using the 5-PL method. Seven measurements were obtained to construct a standard curve. An unpaired t-test was used to test for differences between the pre-challenge and peak cytokine production post-challenge period (3, 7, 14, 21, and 28 days after challenge) for each animal, owing to the high variability in starting concentrations between animals as well as the variability in the peak response for each cytokine post-infection. All sample measurements were repeated in duplicates.