Tumor-Treating Fields Induce RAW264.7 Macrophage Activation Via NK-?B/MAPK Signaling Pathways

Park, Jeong-In; Song, Kyung-Hee; Jung, Seung-Youn; Ahn, Jiyeon; Hwang, Sang-Gu; Kim, Joon; Kim, Eun Ho; Song, Jie-Young

Total RNA was extracted from RAW 264.7 cells using TRIzol reagent (Invitrogen) and reverse transcribed using EcoDry Premix (Clontech Laboratories, Mountain View, California) according to the manufacturer's recommendations. Quantitative reverse transcription polymerase chain reaction experiments were performed using Maxima SYBR Green qPCR Master Mix (Thermo Scientific) in LightCycler 96 Real-time PCR system (Roche Diagnostics, Mannheim, Germany). Using GAPDH as an internal reference, the relative gene expression was calculated based on the DDCt method. The following primer pairs were used: inducible nitric oxide synthase (iNOS), 5 0 -CGA AAC GCT TCA CTT CCA A-3 0 (forward) and 5 0 -TGA GCC TAT ATT GCT GTG GCT-3 0 (reverse); IL-1b, 5 0 -TGA AGG GCT GCT TCC AAA CCT TTG ACC-3 0 (forward) and 5 0 -TCT CCA TTG AGG TGG AGA GCT TTC AGC-3 0 (reverse); TNF-a, 5 0 -ATG AGC ACA GAA AGC ATG ATC CGC-3 0 (forward) and 5 0 -CCA AAG TAG ACC TGC CCG GAC TC-3 0 (reverse); and GAPDH, 5 0 -ACC ACA GTC CAT GCC ATC AC-3 0 (forward) and 5 0 -TCC ACC ACC CTG TTG CTG TA-3 0 (reverse). Experiments were repeated at least 3 times.