Chromo4 rt pcr detection system

Total RNA was extracted using QIAGEN RNeasy® Mini kit (QIAGEN GmbH, Germany), and cDNA was synthesized using Maxime RT PreMix (Bioneer, Daejeon, Korea) according to the manufacturer’s protocol. Quantitative real-time PCR was performed using SYBR® Green Real-Time PCR Master Mixes (Thermo Fisher Scientific, Inc.) and Chromo4 RT-PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer’s instructions. The relative gene expression was analyzed using the critical threshold method in Opticon Monitor software program.

In the present study, 9-HIK was isolated as described previously (13 (link)). Dulbecco's modified Eagle's medium (DMEM), FBS and penicillin-streptomycin were purchased from Hyclone (Cytiva). Reagents, such as LPS, DMSO, Griess reagent, N-acetyl-L-cysteine (NAC, an ROS scavenger), NP40 cell lysis buffer and protease inhibitor cocktail were purchased from Sigma-Aldrich (Merck KGaA). An EZ-Cytox cell viability assay kit was purchased from Daeil Lab Service, Co., Ltd. An RNeasy kit was purchased from Qiagen GmbH. A PrimeScript™ II 1st strand cDNA synthesis kit and TB Green^® premix Ex Taq™ (Tli RNaseH Plus) were obtained from Takara Bio, Inc. A Chromo 4 RT-PCR detection system was purchased from Bio-Rad Laboratories, Inc. Rabbit polyclonal antibodies against β-tubulin (cat. no. SC-9104) and HO-1 (cat. no. SC-10789) were obtained from Santa Cruz Biotechnology, Inc. Rabbit polyclonal antibodies against iNOS (cat. no. 2977S), Nrf2 (cat. no. 12721S) and lamin B (cat. no. 13435S) were purchased from Cell Signaling Technology, Inc. Goat anti-rabbit IgG HRP-conjugated secondary antibody (cat. no. A16110) was supplied by Invitrogen (Thermo Fisher Scientific, Inc.). ELISA kits for IL-6 detection (cat. no. SM6000B) were purchased from R&D Systems, Inc. The ELISA kit for interferon (IFN)-β (cat. no. 42400-2) was obtained from Pestka Biomedical Laboratories, Inc.