Cytokine and Chemokine Quantification by Luminex

Supernatants from CD14⁺ cellular cultures were quantified by multiplex Luminex™ 100 suspension array system (Bio-Plex 200; Bio-Rad Laboratories, Hercules, CA, USA) per the manufacturer’s instructions, to detect the presence and concentrations of cytokines and chemokines commonly produced by monocytes. These methods have been described in detail in previous studies [40 (link)]. Briefly, antibody-coupled beads were added to 25 µL of supernatant and incubated then washed to remove unbound beads. They were next incubated with biotinylated detection antibody followed by the addition of streptavidin–phycoerythrin. Sample concentrations were analyzed by Luminex and concentrations were determined by Bio-Plex Manager software using a standard curve derived from serial dilutions of reference cytokine concentrations included in the assay, with final concentrations calculated using a five-parameter model. Assay limits of detection (LOD) were: GM-CSF (2.3 pg/mL), IL-1β (0.7 pg/mL), IL-6 (0.4 pg/mL), IL-10 (0.3 pg/mL), IL-12(p40) (12.3 pg/mL), IL-12(p70) (0.9 pg/mL), IP-10 (1.3 pg/mL), MCP-1 (1.2 pg/mL), and TNFα (0.2 pg/mL). One-half of the LOD was used to replace values below the limit of detection for statistical analysis.