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

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....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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