Nano glo dual luciferase assay system kit

To evaluate the specificity of mpFN26A[Fluc]/mpGL4.35[Nluc], the sensor system was tested in uniplex and triplex modes, in transfected COS-1 cells exposed to DMSO or 200 μM of clotrimazole, a fungicide ligand of Pregnane X receptor (PXR) [42 (link)]. Luminesc ence activity was measured 24 h later with Nano-Glo^® Dual Luciferase Assay System kit (Promega, Madison, WI, USA). This assay was independently performed three times, with two technical replicates in each assay.

The regulation of the WNT7A promoter mediated by HIF-1α was evaluated with the Nano-Glo^® Dual-Luciferase^® Assay System kit (Promega), which enables a high-throughput analysis of mammalian cells containing the following reporter genes: (a) Nanoluc (pNL1.1empty and pNL1.1WNT7A_promoter) and (b) pGL4.54(luc2/TK), containing the reporter gene Firefly as internal control. Briefly, cells were seeded in a 96-well plate at a concentration of 5 × 10³ cells per well, transfected with the pNL1.1 or pNL1.1WNT7A_promoter (90 ng) plasmids and pGL4.54(luc2/TK; 10 ng) plasmid as internal control, according to ViaFect Transfection Reagent^® protocol (Promega), and then incubated for 24 h. After transfection, cells were exposed for 24 h to hypoxic culture conditions (1% O₂) or to the PHDs inhibitors, IOX2 (Merck) or FG-4592 (Roxadustat, Selleckchem), at their respective IC₅₀ working concentrations. Then, an equal volume of ONE-Glo^TM EX Luciferase Assay Reagent was added to each well, incubated for 30 min, and then the luminescence was analyzed with a Varioskan^TM LUX Multimode Microplate Reader (Thermo Fisher Scientific). To turn off the luminescence of the Firefly luciferase and provide the substrate for the Nanoluc, an equal volume of NanoDLR^TM Stop&Glo^® Reagent was added to the mixture and incubated for 30 min before analysis.