Dual luciferase reporter assay kit

LIF luciferase reporter plasmids were obtained from OBiO Technology (Shanghai, China). For reporter constructs, the 1326-bp promoter region (70/+1255) of the LIF gene was cloned into the pGL4.1 firefly luciferase reporter vector. phRL-TK containing the Renilla luciferase gene was used as an internal standard for transfection efficiency. Cells were seeded into 24-well plates at a density of 1 × 10⁵ cells/well and co-transfected with 500 ng luciferase construct plasmid or an empty reporter vector DNA and 0.05 μg phRL-TK using jetPEI™ transfection reagent for 48 h (Polyplus-transfection). The cells were then lysed, and the luciferase activity was examined with the dual-luciferase reporter assay kit (Transgen Biotech) on a microplate reader with a multi-wavelength measurement system (Synergy™ H1, BioTek Instruments, Inc., Winooski, VT, USA). Luciferase activity was calculated relative to the Renilla luciferase activity.

In order to determine levels of Tet-On 3G system-associated background expression, we used a dual-luciferase system to normalize cell numbers and transfection efficiency. Cells were grown in the absence or presence of doxycycline (50 ng/mL) in 24-well plates. The vectors and internal reference plasmid pxl-BacII-Rluc, which can express Renilla luciferase in sf9 cells, were co-transfected into the cells using Cellfectin II reagent (Invitrogen) according to the manufacturer’s protocol. Relative luminescence was measured at 48 h post-transfection using a Dual Luciferase Reporter assay kit (Transgen). Luminometric analysis was conducted using a PerkinElmer infinite 200 plate reader (Tecan).