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. Prepare a 5 mM carboxyfluorescein succinimidyl ester (CFSE) stock solution by adding 18 µl of DMSO to the vial. 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 u…
Divulgations
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 |
Tags
Citer Cet Article
Killing K562 Cells by Peripheral Blood Mononuclear Cells Exposed to Tobacco Product Preparations. J. Vis. Exp. (Pending Publication), e21645, doi: (2023).