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

Abstract

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

This video demonstrates the effect of tobacco product preparations, TPPs, on cytokine production from human peripheral blood mononuclear cells using flow cytometry. Treatment of the cells with TPP downregulates the production of pro-inflammatory cytokines, which is estimated after lipopolysaccharide stimulation, followed by immunostaining and flow cytometry analysis.

Protocol

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 Generate whole smoke-conditioned medium (WS-CM) as previously described. Prepare WS-CM by passing smoke from four 3R4F reference cigarettes through Roswell Park Memorial Institute (RPMI) 1640 medium without phenol…

Materials

12 X 75 tubes	BD Falcon	352058
15 ml conical tubes	Corning	430790
2 mL Microtubes	Axygen	MCT-150-C-S
3R4F reference cigarettes	Univ. of Kentucky, College of Agriculture	3R4F
50 ml conical tubes	Corning	430828
500 ml bottle	Corning	430282
96-well flat-bottom plate	Termo Nunc	439454
Cell culture hood	Thermo Scientific	1300 Series A2
Centrifuge	Eppendorf	58110R
Cytofix/Cytoperm (Permwash)	BD Biosciences	555028	Flammable
FBS	Sigma-Aldrich	F2442
Filter unit	Nalgene	156-4020
Flow Cytometer	BD Biosciences	FACS Canto II	8 colors, at Ex 405 and Em785.
Flow Cytometer	BD Biosciences	FACS Calibur	4 colors at Ex 495 and Em 785.
Flow cytometry analysis software	Tree Star	FlowJo
Freezing Container	Nalgene	5100-0001	Contains DMSO,  irritant
GogliPlug	BD Biosciences	555029	Carcinogen, Irritant, Corrosive
IFN-γ V-500 Antibody	BD Horizon	561980	skin sensitizer
Isolation Buffer	Isolymph, CTL Scientific Corp.	1114868	Flammable liquid, Irritant
Isopropyl alcohol	Sigma-Aldrich	W292907
L-Glutamine	Gibco Life Technologies	25030-081
LPS	Sigma-Aldrich	L2630
MIP1-α PE Antibody	BD Pharmingen	554730	Acute toxicity, Oral
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
TNF-α Alexa Fluor 488 Antibody	BioLegend	502915
Transfer pipette	Fisher Scientific	13-711-20