Nrf2-Dependent Luciferase Assay Protocol

Above mentioned cell lines were transiently co-transfected with the pGL4.37[luc2P/ARE/Hygro] plasmid and the Renilla plasmid using FHD (Promega, Madison, USA) for Pac2 and ZF4, and XHP (Roche, Mannheim, Germany) for ZFL. Subsequently, after exposure to known Nrf2 inducers or pesticides, Nrf2 induction was analyzed using a Dual-Luciferase® Reporter (DLR™) assay (Promega, Madison, USA). Cells were seeded into white, clear-bottom 96-well plates (Corning, New York, USA) at a density of 10⁴ cellls/well for Pac2, 1.25*10⁴ cells/well for ZF4, and 2.5*10⁴ cells/well for ZFL in 100 µl/well. After 24 h of incubation, cells reached a confluency of about 80%. Transient transfection using FHD or XHP, respectively was conducted in a 2 µg reagent to 1.2 µg DNA (0.9 µg reporter plasmid, 0.3 µg control plasmid) ratio, according to the manufacturer’s instructions. After 24 h of post-transfection incubation, cells were exposed to postulated Nrf2 inducers or pesticides. Prepared stock solutions (see section “Chemicals”) were further diluted using the cell type specific nutrition medium (see section “Cell culture”) supplemented with 5‰ EtOH as a solvent. Seeded cells on 96-well plates were either exposed in technical quadruplicates to increasing concentrations (0.1 µM, 1 µM, 10 µM, 100 µM) of postulated Nrf2 inducers tBHQ, SFN, and H₂O₂ or to increasing concentrations (6.25 µM, 12.5 µM, 25 µM, 50 µM, 100 µM) of the pesticides atrazine, deltamethrin, diazinon, diuron, metazachlor, and terbutylazine. Double quadruplicates of 5‰ EtOH solvent-nutrition medium were used as controls. In parallel with the oxidative stress exposure, increasing DMSO concentrations (1%, 4%, 7%, and 10% v/v) were tested as a cytotoxic control. After 24 h incubation, cells were lysed and quantitative Nrf2-dependent luminescence was measured via Dual-Luciferase® Reporter assay according to the manufacturer’s protocol using an auto-injecting Infinite M1000 microplate reader (Tecan, Männedorf, Switzerland). The luciferase activity was expressed as fold change compared to the controls.