Transient Transfection Assay for Influenza DI RNA

293T cells were seeded in 12 well plates at a concentration of 2 × 10⁵ cells/well. Cells were transfected following an established protocol^{15 (link)} Briefly, cells were cotransfected with pCAGGS plasmids encoding PB1 (10 ng), PB2 (10 ng), PA (10 ng), NP (100 ng), reporter segment encoding firefly luciferase (50 ng) and plasmid encoding a DI RNA or an empty plasmid (concentrations indicated in figures). Cells were washed 6 h post transfection and fresh DMEM was added. After 24 h post transfection, cells were harvested and firefly luciferase activity was measured by using a Beetle-Juice Luciferase substrate (PJK). The values were recorded on a Plate Chameleon V reader (Hidex) using Microwin 2000 software.