Bio plex reader

For evaluation of fecal Ig’s, feces were collected, weighed, and resuspended in 1 ml PBS containing 0.1% sodium azide and protein inhibitors. After overnight incubation, fecal pellets were centrifuged at 10,000 g for 15 min, and supernatants were collected. Fecal suspensions were stored at −20°C. Levels of fecal IgA and IgM were measured by a mouse IgA/IgM ELISA quantitation set (Bethyl Laboratories, Inc.) according to the manufacturer’s instructions. Serum levels of IgG1, IgA, and IgM were measured by a multiplex assay kit (Beadlyte Mouse Immunoglobulin Isotyping kit; EMD Millipore) according to the manufacturer’s instructions and run using a Bio-Plex reader (Bio-Rad Laboratories). Levels of IgE were measured in sera by ELISA assay (BD). Cytokine levels in sera were measured using a specific ELISA kit for mouse IFN-γ and TNF (R&D Systems). For cytokine determination, colonic tissues were incubated at 37°C for 48 h. Cultured supernatants were collected after 48 h, and cytokine concentrations were determined by ELISA (R&D Systems) and normalized for tissue weight.

Sputum matrix metalloproteinase (MMP)-9 level was measured using Luminex multiplex assay (Bio-Rad, USA) [13 (link)] with the Bio-Plex Reader (Bio-Rad). The lower and upper bounds of detection were 0.14 and 302.50 ng·mL⁻¹, respectively, for MMP-9. Free neutrophil elastase (NE) activity was quantified in sputum supernatant using an NE activity assay kit according to the manufacturer's instructions (Cayman Chemicals, USA). Measurements were performed in duplicate and the mean was included for further analysis.