Quantification of Inflammatory Mediators in Infected Human Tissue-Derived Cells
Transcrição
During an infection, activated lymphocytes release inflammatory mediators — cytokines — to elicit an immune response.
To quantify inflammatory mediators in an infected tissue, begin with a cell suspension containing human lung-tissue-derived immune cells.
Incubate the cells with Haemophilus influenzae — a respiratory pathogenic bacterium. The bacteria's surface antigens interact with pattern recognition receptors on phagocytic immune cells. This initiates bacterium internalization, processing into smaller peptides, and the presentation of the peptide by the major histocompatibility complex molecule.
The antigen-MHC-II molecule on phagocytic cells binds to T lymphocytes through the T cell receptors, along with co-stimulatory receptor interactions, resulting in T lymphocyte activation and the production of pro-inflammatory cytokines.
Treat the cells with Golgi blockers, impairing cytokine release — causing their accumulation inside the cells. Wash the cells with a blocking buffer to block non-specific binding sites.
Stain the cell suspension with a mix of fluorophore-labeled antibodies that bind specifically to human T lymphocyte-surface markers, enabling lymphocytes' labeling.
Fix the cells, and permeabilize them with a pore-forming surfactant. Incubate the permeabilized cells with fluorophore-labeled anti-cytokine antibodies, which enter the cells and interact with specific cytokines.
Using flow cytometry, measure the cytokine fluorescence signal within labeled lymphocytes, correlating with inflammatory mediator production due to bacterial infection.
