2.13. Meso Scale Discovery (MSD) assay

Spleen cells (10⁶ cells/well) were stimulated with 5 µg/ml unmodified LYS in sterile-Nunc U-bottom Nunclon Delta 96-well surface plates (Thermo Fisher Scientific), and the supernatants were harvested after 72 h incubation at 37 °C/ 5% CO₂. The concentrations of secreted cytokines in the supernatants were measured using the MSD® U-Plex® Biomarker Group 1 (mouse) Multiplex Assays (Meso Scale Diagnostics, Rockville, MD, USA). A plate used to measure IFN-γ, IL-5, IL-10 and IL-17 was prepared by mixing each linker with its corresponding biotinylated antibody for 1 h at room temperature (RT) with stop solution (MSD® U-Plex®) added during the last 30 min of the incubation period. The plate was coated with the linker-antibody solution for 1 h with shaking at RT. A volume of 25 µl of the stimulated spleen cells was added to each well containing 25 µl Diluent 41, and the plate was incubated for 1 h at RT. Calibrators 5 and 7 was included on the plate as standards. After washing the plate with PBS supplemented with 0.05% (v/v) Tween 20, detection antibody solution diluted in Diluent 45 was added to each well, and the plate was incubated for 1 h with shaking. Read buffer T was added to the plate before measuring the electrochemiluminescence using a Meso Sector Imager 2400 plate reader equipped with Discovery Workbench 4.0. MSD software (Meso Scale Diagnostics).