Luminex 100 suspension array system

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.

We collected peripheral blood from all subjects in acid-citrate dextrose Vacutainers (BD Biosciences, San Jose, CA, USA). Plasma was obtained from blood by centrifugation at 2100 rpm for 10 min. Then, the plasma was separated, aliquoted, and stored at − 80 °C for further cytokine analysis. A panel of four cytokines (MMP-1, MMP-3, osteopontin, and pentraxin-3) was measured in the plasma for Bio-plex Pro human inflammation panel (Bio-Rad, 17008653, Hercules, CA, USA) detection. We ran one sample per subject at one time, in line with the operating protocol. First, we incubated a 25-μl plasma sample with antibody-coupled fluorescent beads. Second, we washed and incubated the sample with biotinylated detection antibodies detected by streptavidin–phycoerythrin. Third, a flow-based Luminex 100 suspension array system (Bio-plex 200; Bio-Rad Laboratories, Inc.) was used to analyze the beads. To determine the sample concentration, standard curves were generated by Bio-plex Manager software, and in the kit, the reference cytokines were provided by the manufacturer. Other inflammation cytokines (tumour necrosis superfamily member 13B [TNFSF/BAFF], IL-29/interferon [IFN]-λ2, IL-32, TWEAK/TNFSF12, and sCD30/TNFRSF8, IFN-γ, IL-10, IL-12p70, IL-17A, IL-1β, IL-2, IL-4, IL-6, and TNF-α) were assessed with the same method in our previous research [11 (link)].