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

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)

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…

Declarações

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
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

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Analyzing the Effect of Tobacco Product Preparations on Cytokine Production via Intracellular Staining and Flow Cytometry. J. Vis. Exp. (Pending Publication), e21618, doi: (2023).

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