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…
開示
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 |
