Optogenetic Regulation of Luciferase

HEK 293T cells were plated at 0.5 × 10⁵ cells/well in 24 well plates (Corning) coated with poly-ornithine (Sigma-Aldrich). The following day, the cells were transfected with cDNAs encoding PA-Cre along with Firefly Luc reporter and Renilla Luc using X-treameGENE9 reagent (Sigma-Aldrich). The standard transfection ratio of PA-Cre:double-flox-inverted-FLuc:HSV-TK-Renilla Luc was as follows: 1:9:0.1. The cells were then exposed to blue light (total 12 h, 20 s light/60 s dark cycle, homemade device^{13 (link)}) 36 h after transfection at 37 °C in a CO₂ incubator (Thermo-Fisher). In case of tamoxifen-treated Mock or CreERT2, the cells were treated with 1 μM tamoxifen (Sigma-Aldrich) 36 h after transfection. Luc activity levels were assayed right after illumination was terminated using Dual Luciferase assay kit and Veritas 96-well luminometer (Promega) following the manufacturer’s instructions.