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

Published: September 29, 2023

Abstract

Source: Arimilli, S. et al., Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations. J. Vis. Exp. (2015)

In this video, we study the effect of a whole smoke-conditioned medium, a combustible tobacco product preparation, on K562 cell killing by effector peripheral blood mononuclear cells.

Protocol

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.

Disclosures

The authors have nothing to disclose.

Materials

12 X 75 tubes BD Falcon 352058
15 ml conical tubes Corning 430790
2 mL Microtubes Axygen MCT-150-C-S
50 ml conical tubes Corning 430828
500 ml bottle Corning 430282
7AAD BD Pharmingen 559925
96-well flat bottom plate Termo Nunc 439454
96-well round bottom plates BD Falcon 353077
Cell culture hood Thermo Scientific 1300 Series A2
Centrifuge Eppendorf 58110R
CFSE Molecular Probes Life Technologies C34554
Cluster tubes Corning 4401 Harmful if swallowed, carcinogen
DMSO (Dimethyl sulfoxide ) Sigma-Aldrich D8418
DPBS Lonza 17-512F
FBS Sigma-Aldrich F2442
Filter unit Nalgene 156-4020
Flow Cytometer BD Biosciences FACS Canto II 8 colors at Ex 405 nm and Em785 nm.
Flow Cytometer BD Biosciences FACS Calibur 4 colors at Ex 495 nm and Em 785 nm.
Flow cytometry analysis software Tree Star FlowJo
Nicotine Sigma-Aldrich N3876 Acute toxicity, Environmental hazard
Parafilm Bemis "M"
Paraformaldehyde Sigma-Aldrich P6148 Flammable, Skin irritation
Pen/strep Gibco Life Technologies 15140-122
RPMI 1640 Gibco Life Technologies 11875-093
Running buffer MACS Running Buffer, Miltenyi Biotech 130-091-221
Transfer pipette Fisher Scientific 13-711-20
Tris Base Sigma-Aldrich T1503

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Cite This Article
Killing K562 Cells by Peripheral Blood Mononuclear Cells Exposed to Tobacco Product Preparations. J. Vis. Exp. (Pending Publication), e21645, doi: (2023).

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