Luminex multiplex assay

Concentrations of six cytokines (IL-1β, IL-6, IL-17, TNF-α, IFN-γ, and MCP-1) in rat serum were determined using the Luminex Multiplex assay (Catalog No. 12005641; Bio-Rad, Hercules, CA) according to the manufacturer’s protocol. All multiplexing assays were performed on the Bio-Plex MAGPIX Multiplex reader system (Catalog No. 171015001; Bio-Rad). Briefly, serum samples were incubated with capture antibody-coupled magnetic beads. After three washes in a Tecan washing station, the samples were incubated with the biotinylated detection antibody. Each captured cytokine was detected by the addition of streptavidin–phycoerythrin. The standard curve was utilized to convert optical density values into cytokine concentrations (pg/ml).

Antigen-specific T cells were stimulated with peptide-pulsed autologous B-LCLs in an effector cell to target cell (E:T) ratio of 1:1 or 1:2. For intracellular staining, cells were co-cultured with the presence of anti-CD107a, 0.7 μg/ml monensin (BD Biosciences), 10 μg/ml brefeldin A (BD Biosciences) in R10 medium for 4 h at 37 °C. For supernatant cytokine quantification (Luminex multiplex assay), cells were co-cultured for 24 h and the supernatants were harvested and kept in −80 °C until being analysed using Bio-Plex assay kits (Bio-Rad) on a Luminex machine (Bio-Rad). Negative controls included the supernatant from un-stimulated cells.