Pei 40k

MLVs pseudotyped with various envelope proteins were generated as previously described [5] (link). Briefly, HEK293T cells were co-transfected with three plasmids, pMLV-gag-pol, pQC-Fluc and pCAGGS-SARS2-SFmut-cflag or pCAGGS-VSV-G using PEI 40 K (Polysciences, Inc.), and the medium was refreshed 6 h later. The supernatant containing the pseudotyped virus was harvested 72 h post-transfection and clarified through a 0.45 µm filter. Clarified viral stocks were supplemented with 10 mM HEPES and stored at −80 °C for long-term storage. Pseudotyped viruses were titrated using TCID₅₀ method. Briefly, HEK293T-ACE2 cells were seeded in 96-well plates at 50–60% confluency upon observation the following day. Then, 50 µL of media was removed from each well and replaced with 50 µL of the serially diluted pseudotyped virus or media. The plate was centrifuged at 4 °C, 3000 g for 30 mins (spinoculation) and incubated for 2 h at 37 °C and 5% CO₂. Virus containing media was then aspirated and 100 µL of fresh media with 1 µg/mL puromycin was added to the wells. The plate was further incubated for 48 h at 37 °C and 5% CO₂. Then, 100 µL/well of OneGlo (Promega) was added and luminescent signal was read. The average and 3 standard deviations of the signal level for media only wells were calculated and used as a cutoff. TCID₅₀ was calculated using Reed & Muench Calculator [6] (link).