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Analyzing the Effect of Tobacco Product Preparations on Cytokine Production via Intracellular Staining and Flow Cytometry

Analyzing the Effect of Tobacco Product Preparations on Cytokine Production via Intracellular Staining and Flow Cytometry

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Upon interaction with inflammatory stimulants, immune cells release inflammatory signaling molecules — cytokines — to produce an immune response.

To analyze the effect of tobacco product preparations, TPPs, take a multi-well plate with increasing TPP concentrations. Seed the plate with human peripheral blood mononuclear cells, PBMCs containing immune cells.

Nicotine — an immunosuppressive agent in TPPs — binds to nicotinic acetylcholine receptors, downregulating pro-inflammatory cytokine production. This downregulation is dose-dependent, with higher concentrations having a more significant effect.

Centrifuge to pellet the cells and discard the supernatant containing TPP residues.

Resuspend the cells in a complete medium and add a mix containing lipopolysaccharide — an immune stimulator and GolgiPlug — a protein transport inhibitor.

Lipopolysaccharide molecules interact with immune cells containing toll-like receptor-4, triggering intracellular signaling pathways and the production of pro-inflammatory cytokines. Meanwhile, GolgiPlug molecules block cytokine secretion from the Golgi apparatus, enabling its accumulation.

Fix the cells with a fixative solution and permeabilize them with a pore-forming agent. Overlay the cells with a cocktail of fluorophore-tagged anti-cytokine antibodies, which enter through the pores and bind to respective intracellular cytokines.

Analyze the stained cells by flow cytometry, which measures fluorescence signals.

Determine the level of cytokine production; a dose-dependent reduction indicates the immunosuppressive effect of TPPs.

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