Mouse and human IL-1β DuoSets were obtained from R&D Systems (Minneapolis, MN) and ELISA assays performed according to the manufacturer’s protocol. IL-18 capture and detection antibodies were also obtained from R&D Systems. The IL-18 ELISA, although developed in-house, was run similar to R&D Systems IL-33 DuoSet ELISA with regard to timings, diluents, standard curves, and washes. Lavage fluid samples were assayed without dilution. In vitro media supernatants were diluted (1:5 for mouse cells, 1:100 for human cells) for optimizing the fit to the kit’s standard curve. All plates were read at 450 nm and data expressed as pg/ml.
Cathepsin activity for the first WLL fluid was determined by mixing the following assay components in a 96-well plate using PBS as diluent: first WLL fluid (50 μl), 2 μg Z-LR-AMC (fluorogenic Peptide Substrate, R&D systems, Minneapolis, MN) in a total volume of 150 μl. The assays samples were incubated at 37 °C for 1 hour then fluorescence was measured using a plate reader at 380 nm excitation and 460 nm emission.
Cathepsin activity for the first WLL fluid was determined by mixing the following assay components in a 96-well plate using PBS as diluent: first WLL fluid (50 μl), 2 μg Z-LR-AMC (fluorogenic Peptide Substrate, R&D systems, Minneapolis, MN) in a total volume of 150 μl. The assays samples were incubated at 37 °C for 1 hour then fluorescence was measured using a plate reader at 380 nm excitation and 460 nm emission.