Generating Functional Interferon Lambdas

IFNλs were produced using the protocol described previously, which is capable of generating functional IFNλs (17 (link)). Briefly, IFN expression plasmids were transfected into sub-confluent HEK-293T cell monolayers, which are hyporesponsive to IFNλ signalling due to very low expression of IFNλR1 (15 (link)). Lipofectamine 2000 was used to transfect IFNλ plasmids per manufacturer’s instructions. IFNλs were routinely generated in six-well plates or 10 cm dishes, and 2 and 14 µg of plasmids were used, respectively. Lipofectamine 2000 (2 µl) was used per microgram of plasmid. Plasmids were transfected into cells in Optimem for 16–18 hours, before changing media to growth media (10% FCS) until 2 days posttransfection was reached. The conditioned media were harvested, clarified by centrifugation, aliquoted, and immediately frozen at −80 in. Relative levels of IFNλs were estimated using the extracellular HiBiT split luciferase assay by virtue of their C-terminal HiBiT tag by incubating IFN preparations with assay reagents and measured by manufacturer’s instructions (Nano-Glo HiBiT Extracellular Detection system, Promega) using a luminometer.