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

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.