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

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.