Plate chameleon 5 plate reader

293T were seeded at a cell density of 2 × 10⁵ cells/well in 12-well plates. The following day, the cells were transfected using the calcium phosphate method. The concentrations of plasmids to be transfected were largely adapted from published work [30 (link)]: 10 ng of pCAGGS plasmids encoding viral RNA polymerase proteins (PB2, PB, PA) and 100 ng of plasmid encoding NP were cotransfected with 50 ng of plasmid pPolI-Luc, which encodes the firefly luciferase reporter gene flanked by the noncoding regions of segment 8 of A/WSN/33. Empty plasmid was used to ensure that all transfections were conducted with the same total amount of plasmid DNA. For analysis of functionality of PB2 in 293T cells stably expressing this protein, transfection was carried out as described above but the plasmid encoding PB2 was omitted. As control, the plasmid encoding PB1 was omitted. The cells were washed at 6–8 h after transfection and harvested at 24 h post transfection. Luciferase activities in cell lysates were measured using the Plate Chameleon V plate reader (Hidex) and Microwin 2000 software.