2.5.5. Inflammatory Cytokines
The simultaneous assessment of serum concentrations of IL-2, IL-4, IL-5, IL-8, INF-γ, inducible protein 10 (IP-10), and soluble CD40 ligand (sCD40L) was performed using commercially available multiplex bead-based immunoassay kits (Milliplex MAP Human Cytokine/Chemokine Magnetic Bead Panel, HCYTOMAG-60K, Millipore, Billerica, MA, USA) following manufacturer’s instructions. Briefly, plasma samples (25 μL/well) or standards (25 μL well) were incubated with 25 μL of the pre-mixed bead sets in pre-wetted 96-well microtiter plates at 4 °C overnight. After washing, 25 μL of the fluorescent detection antibody mixture was added for 30 min, and 25 μL of streptavidin-phycoerythrin was added to each well for 30 min at room temperature. A range of 3.2–10,000 pg/mL recombinant cytokines was used to establish standard curves and maximize the assay’s sensitivity and dynamic range. Cytokine levels were determined using a Luminex IS 100 (Luminex, Austin, TX, USA).