Luminex Analysis

The levels of cytokines and chemokines (IL-1β, IL-6, IL-13, TNFα, Gro/KC, and MCP-1) in BAL fluid were determined using a multiplex assay kit (Lincoplex panel, Linco Research, Inc., St. Charles, MO) according to the manufacturer’s instructions. Briefly, the BALF was filtered to remove cells and debris, then beads were incubated with diluted standards, or BALF overnight followed by a detector antibody cocktail for 60 min each at room temperature. After two washes in PBS supplemented with 0.02% Tween 20, 0.1% BSA, and 0.02% NaN3, the beads were incubated for 30 min with fluorescent dye-conjugated streptavidin. Cytokine levels were measured using a flow cytometer and were analyzed with Flowmetrix software (Luminex, Ausitn, TX). Standard curves for each cytokine and chemokine were generated on a log-log plot for each assay, and the concentrations in each sample were calculated from the corresponding curve-fitting equations.