Quantification of Spike-Pseudotyped Lentiviral Infectivity

Pseudotyped lentiviral vectors were produced as previously described^{32 (link)}. Briefly, HEK293T cells were co-transfected in a 2:1 ration with the pNL4-3-inGluc vector and the spike plasmid of interest using polyethyleneimine transfection (Transporter 5 Transfection Reagent, Polysciences) to produce pseudotyped lentiviral particles. The lentivirus was collected by taking the media of the transfected cells 48 and 72 hours post-transfection. Relative infectivity of the lentivirus was then assessed in both HEK293T-ACE2 and CaLu-3 cells. Gaussia luciferase activity measured at 72 hours post infection for HEK293T and 72 hours for CaLu-3 were used to determine relative infectivity. Gaussia luciferase activity was determined by taking equal volumes of infected cell media and Gaussia luciferase substrate (0.1 M Tris pH 7.4, 0.3 M sodium ascorbate, 10 μM coelenterazine) and combining for an immediate luminescence signal detected by a BioTek Cytation plate reader.