Glomax 20 20 single tube luminometer

Twenty-four hours prior to transfection immortalized baboon central disc TMJ cells were seeded into 24-well plates at 5 × 10⁵ cells per well. The cells were co-transfected at 50% confluency in triplicate with the pGL3+promoter construct, empty pGL3 plasmid, or a positive pGL3 control along with the pRL-TK plasmid, a renilla luciferase reporter vector to serve as a control for transfection efficiency. FuGene 6 (Roche) was used to transfect the cells according to the manufacturer’s protocol. Post transfection, wells were treated with or without 10 nM E2 in phenol red-free DMEM plus 10% charcoal-tripped FBS with media and E2 replaced every 24 h. Cells were lysed at 48 h post-transfection and both the firefly and renilla luciferase activity monitored using the Dual Luciferase Assay kit (DLR; Promega) and GloMax-20/20 Single Tube Luminometer (Promega) per manufacturer’s suggested protocol. Luciferase activity was normalized to renilla luciferase activity and all experiments were performed at least three times independently with triplicate samples for each. Statistical significance was determined using the student’s T test.