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

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. WS-CM Preparation

1. Generate whole smoke-conditioned medium (WS-CM) as previously described.
   1. Prepare WS-CM by passing smoke from four 3R4F reference cigarettes through Roswell Park Memorial Institute (RPMI) 1640 medium without phenol red using the following smoking regimen: 35-60-2, puff volume in ml, puff interval in sec, and puff duration in sec, respectively. Each preparation generates a 20 ml sample.
2. Label tube(s) with date, time completed, and cigarette name and number. Store the 500 µl aliquots at -80 °C immediately after smoking is completed.
3. Analyze aliquots of frozen WS-CM to determine the levels of nicotine, tobacco-specific nitrosamines, and polycyclic aromatic hydrocarbons as previously described.

2. Isolation of peripheral blood mononuclear cells, PBMCs

1. Collect fresh blood from healthy donors (who are non-consumers of tobacco products) Isolate PBMCs from fresh blood as described below.
   1. Prior to the arrival of the blood bag, have a 500 ml bottle, scissors, isolation buffer, and Dulbecco's phosphate buffered saline (DPBS) (RT) ready in a biosafety level 2 (BSL-2) cell culture hood. The isolation buffer must be protected from light.
   2. Hold the blood bag upside down and cut the tube just below where it has been clamped leaving at least 3 cm of tubing.
   3. Remove the cap from the 500 ml bottle and hold the tube above the bottle opening. Pick up the blood bag and invert it to allow the blood to flow freely from the bag into the bottle until the bag is empty. Allow the blood to flow onto the inside wall of the bottle versus straight down to avoid creating bubbles.
   4. Pour isolation buffer into the bottle at a 1:5 ratio of isolation buffer to blood. Cap the bottle tightly and gently invert it, end-over-end, 10 times. Leave the bottle in the cell culture hood to incubate with lights off, for 1 hr at RT.
   5. A light, straw-colored layer will build up above the blood. Remove this layer using a 25 ml serological pipette into 50 ml conical tubes. The collected amount may vary from 50 – 300 ml, depending on the subject who donated blood.
   6. Centrifuge the tubes at 200 x g for 10 min at RT.
   7. Aspirate the translucent supernatant, leaving the dark blood-colored pellet. The pellet will be loose but viscous.
   8. Pipette 3 ml of isolation buffer into 15 ml conical tubes.
   9. To the resuspended pellet, add 20 ml of DPBS for every 50 ml of straw-colored liquid collected in step 2.1.5. Vortex to mix thoroughly, and consolidate the resuspended liquid from multiple tubes. This liquid contains suspended blood cells.
   10. With a transfer pipet, transfer 5 ml of the suspended blood cells onto 3 ml of isolation buffer in step 2.1.8. Tilt the 15 ml conical tube that contains the cell suspension slowly and gently to create two separate layers. Centrifuge the tubes at 400 x g for 40 min at RT with minimal acceleration and without brake.
   11. Use a transfer pipet to remove the resulting cloudy middle layer (buffy coat) containing PBMCs into a 50 ml conical tube. Avoid drawing other clear layers below it. Transfer no more than 25 ml into each 50 ml conical tube.
   12. Add 25 ml of cold running buffer (or more to fill the entire remaining volume of the 50 ml conical tube) to wash the cells. Centrifuge the cells at 400 x g for 10 min at 4 °C.
   13. Resuspend the pellet with 10 ml running buffer. This contains PBMCs. Count the cells and use them immediately or place them on ice for freezing.

3. Freezing and Thawing the PBMCs

1. Centrifuge the eripheral blood mononuclear cells (PBMCs) that were collected in step 2.1.13 for 10 min at 400 x g.
2. Fill the freezing container with isopropyl alcohol per the manufacturer's instructions.
   CAUTION: Isopropyl alcohol is flammable and acutely toxic.
3. Resuspend the pellet with RPMI 1640 medium (4 °C) containing 20% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO). This is RPMI 1640 freezing medium. Resuspend the pellet with an amount of freezing medium that will result in a suspension having about 5 x 10⁷ cells/ml. The number of cells available for freezing will vary.
4. Dispense 1 ml aliquots of cell suspension into 2 ml cryotubes and place the cryotubes in the freezing container. Place the freezing container with cryotubes in a freezer at -80 °C to store O/N and then remove the cryotubes and transfer to store in a cryogenic freezer between -150 °C to -190 °C.
5. Remove the cryotube from cryogenic storage and thaw it rapidly with gentle agitation in a water bath at 37 °C.
6. Immediately transfer the thawed PBMCs in the cryotube into a 15 ml conical tube with 10 ml RPMI 1640 complete medium (4 °C) containing 10% FBS, 1% Pen/Strep and 1% L-glutamine. The contents of the thawed cryotube should be transferred as soon as possible to obtain maximal cell viability.
7. Centrifuge the PBMCs at 400 x g for 10 min.
8. Resuspend the pellet with 5 ml RPMI 1640 complete medium and count the cells.
9. Measure cell viability by established methods such as the trypan blue exclusion method. Generally, cell viability with this method is about 90 – 95%. The PBMCs are now ready to use in experiments.

4. Intracellular Staining and Flow Cytometry

NOTE: Dilutions listed here are for the purpose of this study. The dilutions can be adjusted accordingly.

1. Dilute WS-CM in a 96-well plate using RPMI complete medium to a total volume of 100 µl/well at the concentration of 0.3, 1.56, 3, and 5 µg/ml of equi-nicotine units.
2. In the same plate, dilute nicotine to the following concentrations: 2, 10, 50, 100, 500, 2000, and 4000 µg/ml.
   CAUTION: Nicotine is acutely toxic and environmentally hazardous.
3. Add 100 µl of PBMCs suspended in RPMI complete medium at a concentration of 1 × 10⁶ cells/well. The total volume of cells plus WS-CM or nicotine will be 200 µl/well.
4. Cover the plate and incubate for 3 hr at 37 °C and 5% CO₂.
5. Wash the cells at RT by centrifuging at 300 × g for 3 min, aspirating the supernatant, vortexing the bottom of the plate with the plate covered and finally resuspending cells with 200 µl of ice-cold running buffer, and repeat the washing step one more time.
6. Add 200 µl of RPMI complete medium to the plate, and repeat the wash step one more time.
7. Prepare the working concentrations of 2 µl/ml GolgiPlug and 10 µg/ml LPS using RPMI complete medium and add 200 µl to each well.
8. Incubate the plate for 6 hr at 37 °C and 5% CO₂.
9. At the end of step 9.8, wash the cells with running buffer (4 °C) and spin at 300 x g for 3 min at 4 °C.
10. Add 100 µl of Cytofix to each well and incubate for 20 min at 4 °C.
11. Wash the cells 3 times as described in step 9.9 with 1x Permwash (4 °C) at 300 x g for 3 min at 4 °C.
12. Add 45 µL of 1x Cytoperm to each well followed by 5 µl of each of one of the following antibodies to each well: TNF-α-Alexa Fluor 488, IFN-γ V500, MIP-1α PE. Incubate at 4 °C for 30 min.
13. Wash the cells two times as described in step 9.9 with 1x Permwash (4 °C) and one time with running buffer (4 °C) at 300 x g for 3 min at 4 °C.
14. Resuspend the cells with 200 µl of 2% paraformaldehyde (4 °C). Transfer the cells into 12 × 75 mm tubes, and analyze the samples on a flow cytometer.
    CAUTION: Paraformaldehyde is corrosive, acutely toxic, and a health hazard.