Twinlite firefly and renilla luciferase reporter gene assay system

HEK293 cells were grown on 96-well plates and transfected at 70–80% confluency with promoter-luciferase constructs (20 ng/well), with RIG-I pathway expression plasmids (30 ng/well, except 3 ng/well for IKKε), and with ZIKV or HCV NS3/4A expression constructs (3–30 ng/well). RSV-Renilla (50 ng/well) was included as an internal control. Cells were harvested at indicated time points for Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) or for twinlite Firefly and Renilla Luciferase Reporter Gene Assay System (Perkin Elmer, Waltham, MA, USA) according to the manufacturer’s instructions. The firefly luciferase results were normalized with the values of Renilla luciferase. All experiments were done in triplicates and repeated at least three times.

Stable immortalized HEK 293T, HEK 293-hTLR2/6, and HEK 293/hTLR4A-MD-2-CD14 cells (InvivoGen, San Diego, CA, USA) were plated at 3 × 10⁶ cells in 6-well plates growing at 37°C in DMEM culture medium supplemented with 5% FBS, 2 mM L-glutamine, 100 U/ml gentamycin, 0.01% pyruvate, and 0.4 mM non-essential amino acids. Then, 24 h later, cells were cotransfected with the pNF3ConA Luc [nuclear factor (NF)-κB] Firefly reporter construct and the thymidine kinase promoter-Renilla reporter plasmid (100:1 ratio) using Metafectene PRO (Biontex, Plannegg, Germany) (19 (link)). Transfection medium was changed after 24 h, and cells were seeded at 1.3 × 10⁴ cells per well in 96-well plates. Then, 24 h later, ligands were added. Activities of Firefly and Renilla luciferases were measured 24 h after using TwinLite Firefly and Renilla Luciferase Reporter Gene Assay System (PerkinElmer) in Fluo Star Optima (BMG) plate reader (three replicates per condition). All ratios were compared with the control condition (non-stimulated cells). We used FSL-1 (InvivoGen), TNF-α (InvivoGen), and LPS purified from E. coli (Sigma) and O. intermedium LPS.

NIH-3T3 cells were transfected in Opti-MEM (Invitrogen) using lipofectamine 2000 (Invitrogen) with 75 ng pGL3(BRE)2-luc, 30 ng pRL-TKluc and 5 ng of plasmids encoding either HA-tagged WT or generated mutants for ALK1. Five hours post transfection, cells were stimulated with or without recombinant human BMP10 (100 pg/mL) for 18 h. Firefly and renilla luciferase activities were sequentially measured with Twinlite Firefly and Renilla Luciferase Reporter Gene Assay System (Perkin Elmer) using the TECAN SPARK multimode microplate reader (Thermofisher). Final luciferase activities were reported as folds of firefly luciferase activities normalized to renilla luciferase activities.