Pseudotyped Virus Luciferase Assay

The 50–92 HA pseudotyped viruses (PVs) encoding firefly luciferase were generated in HEK 293 T cells and treated with exogenous NA (Sigma Aldrich) as previously described [32 (link), 33 (link)]. Then 50 µl of PV stock was mixed gently with an equal volume of pre-warmed MES pH buffer at the indicated pH in 1.5 ml Eppendorf tubes and incubated at 37 °C for 5 min before inoculation of 25 µl per well of confluent HEK 293 T cells in a white bottomed 96-well plate. After 24 hours cell media was removed, and cells were lysed in passive lysis buffer (Promega) before quantification using a firefly Luciferase Assay System (Promega) and a FLUOstar Omega plate reader (BMG Labtech).