Divide the peripheral blood sample into aliquots for control and antigen stimulation. Add co-stimulatory antibodies to both samples, then add the non-typeable H. influenzae to the antigen sample, and incubate at 37 degrees Celsius and 5% carbon dioxide for 1 hour.
Next, add the Golgi-blocking agent Brefeldin A to the samples, and incubate them for another five hours. After lysing the erythrocytes as demonstrated previously, fix the leukocytes using 500 microliters of 1% to 2% paraformaldehyde for 1 hour. Count the cells using a hemocytometer, then permeabilize 1 million cells with 100 microliters of 0.1% saponin for 15 minutes.
Next, incubate the cells with appropriate fluorescent-labeled antibodies. Wash the cells, then analyze them using a flow cytometer. Determine the proportion of antigen-responding cells by gating the relevant lymphocyte populations. Perform background staining on non-stimulated cells for all the cytokines to be analyzed.
