Determination of Plasma and Peripheral Blood Mononuclear Cells (PBMC)- or Spleen Mononuclear Cells (SMC)-Derived Production of the Pro-Inflammatory Interferon (IFN)-γ, Interleukin (IL)-6 and Anti-Inflammatory Interleukin (IL)-10

Peripheral blood mononuclear cells (PBMC) and spleen mononuclear cells (SMC) were examined for their ability to produce pro-inflammatory IFN-γ, IL-6 and anti-inflammatory IL-10 in response to concanavalin A (Con-A) stimulation according to the procedure described previously.⁴⁰ Briefly, PBMC and SMC were seeded at a concentration of 4×10⁶ cells/mL in 24-well Corning tissue culture plates, and then stimulated with a Con-A solution (2.5 μg/mL) or remained non-stimulated (control). The PBMC and SMC were incubated in 37°C. Cell-free supernatants were collected 48 h (for IL-6) and 72 h (for IFN- γ and IL-10) later and stored at −80°C until assayed.

The IFN-γ, IL-6 and IL-10 concentrations in the supernatants and plasma were determined by enzyme-linked immunoassay (ELISA) using a commercially available kit (Rat-IFN-γ and Rat-IL-4 ELISA kits R&D, USA) according to the manufacture’s instructions and our previous study.⁴⁰ Briefly, 50 µL of standards or samples were dispensed into 96 wells coated with rat IFN-γ, IL-6 and IL-10 antibody, respectively, and incubated for 2 h (IFN-γ) or 1 h (IL-4) at room temperature. After extensive washing, 100 µL of the biotinylated anti-IFN-γ or anti-IL-4 were added to each well, and the plates were incubated for 30 min (IFN-γ) or 1h (IL-4) at room temperature. The wells were again washed 3 times, 100 µL of Streptavidin-HRP was added and incubation was carried out for 30 min. 3.3´,5,5´-tetramethylbenzidine (TMB) (100 μL/well) was used as the chromogen for the colorimetric assay. The reaction was stopped after 10 min by adding a 100 µL/well of stop solution and the absorbance was determined using the DTX 880 Multimode Detector (Beckman Coulter, USA) system set to 450 nm. Cytokine concentrations were calculated based on the standard curve generated by Beckman Coulter´s Biomek software program, based on the absorbance of standard samples. The sensitivity of detection was 2 pg/mL for IFN-γ, 16 pg/mL for IL-6 and 3 pg/mL for IL-10.