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.

Before beginning the staining procedure, dilute whole smoke-conditioned medium, and nicotine at the listed concentrations in RPMI complete medium to a total volume of 100 microliters per well in a 96-well plate. Then, add 100 microliters of PBMCs suspended in complete medium at a concentration of 1 times 10 to the sixth cells per well, for a total volume of 200 microliters per well. Next, cover the plate, and incubate it at 37 degrees Celsius and 5% CO₂.

After three hours, wash the cells, and aspirate the supernatant. Then, cover the plate again, and vortex it to dissociate the pellets. Wash the cells two more times, once in ice-cold running buffer, and once in complete medium.

After the second wash, treat them with 200 microliters per well of complete medium, supplemented with GolgiPlug and LPS for 6 hours at 37 degrees Celsius and 5% CO₂. Then, wash the cells with ice-cold running buffer, and treat them with 100 microliters of Cytofix per well at 4 degrees Celsius. After 20 minutes, wash the cells three times with ice-cold permwash.

Then, add 45 microliters of Cytoperm to each well, followed by 5 microliters of each of the listed antibodies. After 30 minutes at 4 degrees Celsius, wash the cells three times, and fix them in 200 microliters of ice-cold 2% paraformaldehyde. Then, transfer the cells into 12 by 75-millimeter tubes, and analyze the samples on a flow cytometer.