This study outlines a method for stimulating lymphocytes using Haemophilus influenzae. It details the process of antigen presentation and T lymphocyte activation through specific cellular interactions.
To stimulate lymphocytes via Haemophilus influenzae — a pathogenic bacterium, take a suspension of isolated peripheral blood mononuclear cells, or PBMCs, including lymphocytes and antigen-presenting cells or APCs.
Add a bacterial suspension and incubate at physiological temperature.
Pattern recognition receptors on the APCs bind to specific components of the bacterial cells, triggering phagocytosis. The phagosome — containing internalized bacteria — fuses with the lysosome, causing bacterial degradation and processing into peptide fragments.
Major histocompatibility complex or MHC molecules bind to the peptide fragments and present them on the cell surface as antigens. T lymphocytes bind to the presented antigen via the T cell receptor.
Add co-stimulatory antibodies that bind to specific surface receptors on T lymphocytes. The synergistic binding induces downstream signaling, activating T lymphocyte cytokine production.
Add a Golgi-blocking compound — blocking vesicular protein transport from the endoplasmic reticulum to the Golgi — to inhibit cytokine secretion from T lymphocytes, facilitating intracellular cytokine accumulation.
Add a fixative to preserve cellular morphology and detergent to permeabilize the cell membrane. Add a cocktail of fluorescently labeled antibodies, including antibodies binding to the T lymphocyte-specific surface markers — labeling the cell surface — and cytokine-specific antibodies that penetrate inside the cell to bind intracellular cytokines.
Perform flow cytometry to detect cells with dual fluorescence — indicating cytokine production by stimulated T lymphocytes.
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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