Quantification of Inflammatory Mediators in Infected Human Tissue-Derived Cells

Slice about 20 to 40 grams of the lobectomy sample into 3 to 5-cubic-millimeter sections. Place them inside a sterile 50-microliter chamber, and mechanically fragment the tissue using an appropriate disaggregator. After tissue disaggregation, lyse the red blood cells as demonstrated and resuspend the cells in sterile RPMI. Then, filter the cells through a 100-micrometer sterile nylon mesh, and count the number of viable cells using the trypan blue exclusion method.

For the infection assay, resuspend the lung cells in RPMI to a final concentration of 4 million cells per milliliter per tube. Then, infect the cells at an MOI of 100 bacteria per cell. Loosen the cap half a rotation to allow gas transfer in the tubes. Place the cells in the tube rotator, and incubate them at 37 degrees Celsius, while rotating at 12 RPM. One hour after stimulation, add Brefeldin A to prevent the extracellular export of cytokines, and incubate the cell suspensions for a further 16 to 22 hours with rotation.

The following day, wash the cell suspension with 500 microliters of PBS containing 1% bovine serum albumin and 0.01% sodium azide. Next, stay in the cell suspension for specific human lymphocyte cell surface markers for one hour. Then, wash the cells with PBS and fix and permeabilize them, as demonstrated previously. Next, incubate the cells with intracellular cytokine-staining antibodies for one hour. Then, wash the cells and resuspend them in 100 microliters of PBS, before data acquisition on a flow cytometer.