Quantification of Immune Mediator Levels in Human Mucosal Lining Fluid

For airway immune mediator quantification, place up to 10 samples from negative 80 degrees Celsius storage on ice, and record their identification numbers. When the samples have thawed, immerse both of the filter papers per subject in 150 microliters of freshly prepared buffer per filter paper.

Supplement it with one complete protease inhibitor tablet per 25 milliliters of buffer stock solution. Transfer the samples to a plate shaker at 400 RPM for 5 minutes. Transfer the buffer and filter papers into the cups of individual cellulose acetate tube filters.

Centrifuge the samples to collect the filtered nasal extract at the bottom of the microcentrifuge tubes. Then, remove the cups, and place the tubes on ice. Next, transfer 150-microliter aliquots of the nasal extract into individual wells of low-protein-binding storage plates, and store the plates at negative 80 degrees Celsius until analysis. Then, measure the concentration of the nasal immune mediators of interest by high-sensitivity electrochemiluminescence-based multiplexed array, according to standard assay protocols.