Spectramax 5 spectrophotometer

To measure concentrations of circulating IL-13, plasma from resting and IL33-treated mice were collected at different time points and measured by ELISA using mouse IL-13 antibody pair kit that detect native and recombinant mouse IL-13 with 0.2 pg/mL sensitivity (Abcam, Cat. ab221432). Amounts of IFNγ in blood were analyzed 2 days after Lm infection using 0.2ug/mL anti-IFNγ capture antibody (R4–6A2 Biolegend Cat. 505702) and 0.2ug/mL biotinylated anti-IFNγ detector antibody (XMG1.2 Biolegend Cat. 505804). Overall, plasma, positive controls, and recombinant standard proteins were diluted, added to ELISA plates (Corning, NY USA) pre-coated with capture antibody and incubated for 2 hours at RT or overnight at 4°C. Biotinylated detector antibody was then added and incubated at 37°C for 1 h followed by streptavidin-conjugated HRP (R&D Systems, MN USA) and visualized with slow kinetic-form TMB (Sigma Aldrich, MO USA). The reaction was stopped with 1N H₂SO₄ (Fisher Scientific, MA USA). Absorbance was measured at 450nm and corrected for absorbance at 540nm using a SpectraMax 5 spectrophotometer (Molecular Devices, CA USA). Measurements were performed in duplicate, and the results were averaged.