Encyclopedia of Experiments
An In Vitro Technique to Stimulate Lymphocytes via Pathogenic Bacteria
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Encyclopedia of Experiments An In Vitro Technique to Stimulate Lymphocytes via Pathogenic Bacteria
An In Vitro Technique to Stimulate Lymphocytes via Pathogenic Bacteria

An In Vitro Technique to Stimulate Lymphocytes via Pathogenic Bacteria

Transcription

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.

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