Mouse elisa max standard sets

To analyze the secretion of IL-1β, IL-6 and IL-10, mouse ELISA MAX^™ Standard sets were used (Biolegend, San Diego, CA, USA) and the assays were performed according to the manufacturer’s instruction. For intracellular IRAK1 and TRAF6, mouse IRAK1 and TRAF6 ELISA kit (LSBio, Seattle, WA, USA) were used. In brief, collected supernatant (for the detection of IL-1β, IL-6 and IL-10) or cell lysate (for the detection of IRAK1 and TRAF6) was added into the antibody pre-coated 96-well plates, incubated, and washed with PBS including 0.1% Tween. After reaction with primary antibody and secondary antibody, substrate solution was added and incubated for 15–30 min at 37°C. The stop solution was applied to each well and the optical density (OD value) of each well was immediately determined at 450 nm using a microplate reader (Bio-tek, Winooski, VT, USA).