Production of SARS-CoV-2 S-pseudotyped lentiviral particles

HEK293T cells were cultured in complete growth media (10% FCS, Pen/strep, L-Glutamine in DMEM) until 60 to 70% confluent on a 10 cm dish. The medium was then replaced with Opti-MEM (supplemented with L-Glutamine and 5% FCS, no antibiotics) for two hours. Cells were then transfected with Lipofectamine 3000 according to the manufacturer ́s instructions. Briefly, a15 solution with Lipofectamine 3000 and a solution with a mix of 18 μg of the following plasmids were separately prepared in Opti-MEM: pMDL g/p RRE, pRSV-REV, pLenti-GFP and a plasmid encoding the spike proteins of SARS-CoV-2 or VSV-G. Plasmid ratio for pMDL g/p
RRE, pRSV-REV, the spike protein plasmid and pLenti-GFP is 1:1:1:2, respectively. The P3000 reagent was then added to the mix. After 5 minutes, the DNA mix was added to Lipofectamine20 solution and the mix was left for 20 minutes at room temperature. The mix was then added to the cells for 6 hours. Cell medium was then changed to normal growth medium (without antibiotics). 24 and 48 hours after transfection, the medium was collected, centrifuged at 1000 x g for 5 minutes, and filtered through a 0.45 μm filter. The medium was centrifuged at 65,000 x g with a SW28 rotor for 3 hours over a cushion of 10% sucrose prepared in PBS. The pellet was finally25
resuspended in TBS buffer containing 5% bovine serum albumin overnight at 4°C, aliquoted and frozen at -80°C