Killing K562 Cells by Peripheral Blood Mononuclear Cells Exposed to Tobacco Product Preparations

1. K562 Killing Assay

NOTE: K562 cells should be grown in culture at 37 °C and 5% CO₂ with RPMI complete medium until they reach 80% confluence before the assay.

1. Prepare a 5 mM carboxyfluorescein succinimidyl ester (CFSE) stock solution by adding 18 µl of DMSO to the vial.
2. Dilute WS-CM or nicotine in a 96-well plate using RPMI complete medium to a total volume of 100 µl/well to achieve the desired equi-nicotine units or nicotine concentrations for each well.
   CAUTION: Nicotine is acutely toxic and environmentally hazardous.
3. Add 100 µl of PBMCs into RPMI complete medium at a concentration of 1.5 x 10⁶ cells/well. The total volume of cells with 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 K562 cells by adding 10 ml of DPBS and centrifuge at 400 x g for 8 min at RT.
6. Resuspend the cells with 10 ml of DPBS and count the K562 cells.
7. Prepare a CFSE working solution by adding 1 µl of CFSE stock solution to 1 ml DPBS.
8. Add 1 ml of the CFSE working solution to 1 ml of the K562 cell suspension containing 1 – 2 x 10⁷ cells. Vortex and incubate precisely for 2 min at RT.
9. Immediately add 200 μl of FBS. Vortex and incubate precisely for 2 min at RT.
10. Add 10 ml of RPMI complete medium and centrifuge the tube at 400 x g for 8 min at RT.
11. Remove the RPMI supernatant, and break the pellet, and resuspend the cells with 10 ml of RPMI complete medium, and count the CFSE-labeled K562 cells.
12. Wash the PBMCs by centrifuging the plate at 300 x g for 3 min at RT. Aspirate the supernatant by decanting the liquid. Replace the cover and vortex the bottom of the plate. Add 200 µl of RPMI complete medium and repeat the washing step one more time.
13. Add CFSE-labeled K562 cells at a ratio of 1:15 (100,000 K562s:1.5 x 10⁶ PBMCs) to each sample well of the PBMC plate and further incubate for 5 hr at 37 °C and 5% CO₂.
14. Wash the cell mix by centrifuging at 300 x g for 3 min at RT. Aspirate the supernatant by discarding the liquid. Place the cover and vortex the bottom of the plate.
15. Add 200 µl of running buffer and repeat the washing step described in step 14 one more time.
16. Add 95 µl of running buffer followed by 5 µl of 7AAD to each well for a total volume of 100 µl/well. Incubate the plate in the dark at RT for 15 min.
17. Add 100 µl of running buffer to each well and transfer the entire volume of cell suspension from each well of the plate to the cluster tubes and acquire the samples on the flow cytometer.
18. Determine the percentage of 7AAD-positive and CFSE-positive cells using flow cytometry analysis software.