Prl tk vector encoding renilla luciferase

Cells were seeded at a density of 30,000 cells per cm² in 24-well plates in 1 mL of DMEM/F12 medium supplemented with 10% FBS without antibiotics. Cells were then transiently transfected with pNFκB-luc reporter construct (Stratagene, San Diego, CA, USA) and pRL-TK vector encoding Renilla luciferase (Promega). Transfections were performed in a final volume of 500 μL of Opti-MEM medium (Thermo Fisher Scientific) with 50 ng/mL of pRL-TK and 200 ng/mL of pNFκB-luc plasmids, and with 1 μL of Lipofectamine 2000 (Thermo Fisher Scientific). After 6 h, the medium was changed to DMEM-F12 (supplemented with antibiotics and 10% FBS), and 24 h later, the treatments were performed. After incubation, cells were lysed in passive lysis buffer (Promega) and luciferase activity was determined with Dual-Luciferase Reporter Assay (Promega), using LM-01T luminometer (Immunotech, Prague, Czech Republic). The Renilla luciferase activity was used for normalization of the firefly luciferase activity.